Anti-integrin antibodies, compositions, methods and uses

ABSTRACT

The present invention relates to at least one novel anti-alpha-V subunit antibodies, including isolated nucleic acids that encode at least one anti-alpha-V subunit antibody, alpha-V subunit, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/598,411, filed 13 Nov. 2006, now issued under U.S. Pat. No.7,550,142, which is a divisional of U.S. application Ser. No. 10/720,323filed Nov. 24, 2003, now issued under U.S. Pat. No. 7,163,681, which isa continuation-in-part of U.S. application Ser. No. 09/920,267, filed 1Aug. 2001 now U.S. Pat. No. 7,288,390, which claims priority to U.S.provisional application 60/223,363 filed 7 Aug. 2000, each of which areentirely incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies which bind to the alpha-Vsubunit of the integrin family of cell adhesion receptors, includingspecified portions or variants thereof. The antibodies of the inventionare specific for at least one alpha-V subunit of a heterodimericintegrin receptor, such as an alpha-V-beta-1, alpha-V-beta-3,alpha-V-beta-5, alpha-V-beta-6, or alpha V-beta-8 heterodimeric integrinprotein or fragment thereof. The invention also relates to nucleic acidsencoding such anti-alpha-V subunit antibodies, complementary nucleicacids, vectors, host cells, and methods of making and using thereof,including therapeutic formulations, administration and devices.

2. Related Art

Integrins are a superfamily of cell adhesion receptors, which exist asheterodimeric transmembrane glycoproteins. They are part of a largefamily of cell adhesion receptors which are involved incell-extracellular matrix and cell-cell interactions. Integrins playcritical roles in cell adhesion to the extracellular matrix (ECM) which,in turn, mediates cell survival, proliferation and migration throughintracellular signaling. The receptors consist of two subunits that arenon-covalently bound. Those subunits are called alpha and beta. Thealpha subunits all have some homology to each other, as do the betasubunits. The receptors always contain one alpha chain and one betachain and are thus called heterodimeric. Both of the subunits contributeto the binding of ligand. Eighteen alpha subunits and eight betasubunits have been identified, which heterodimerize to form at least 24distinct integrin receptors.

Among the variety of alpha chain subunits is a protein chain referred toas alpha V. The ITAGV gene encodes integrin alpha chain V (alphaV). TheI-domain containing integrin alpha V undergoes post-translationalcleavage to yield disulfide-linked heavy and light chains, that combinewith multiple integrin beta chains to form different integrins.Alternative splicing of the gene yields 7 different transcripts; a, b,c, e, f, h, j altogether encoding 6 different protein isoforms ofalphaV. Among the known associating beta chains (beta chains 1, 3, 5, 6,and 8; ‘ITGB1’, ‘ITGB3’, ‘ITGB5’, ‘ITGB6’, and ‘ITGB8’), each caninteract with extracellular matrix ligands. The alpha V beta 3 integrin,perhaps the most studied of these, is referred to as the vitronectinreceptor (VNR). In addition to providing for cell attachment to othercells or to extracellular proteins such as vitronectin (alphaVbeta3) andfibronectin (alphaVbeta6), the integrins are capable of intracellularsignaling which provides clues for cell migration and secretion of orelaboration of other proteins involved in cell motility and invasion andangiogenesis. The alpha V integrin subfamily of integrins recognize theligand motif arg-gly-asp (RGD) present in fibronection, vitronection,VonWillebrand factor, and fibrinogen.

It has been established that integrins which are alpha-V containingheterodimers, particularly alpha-V/beta-6, the receptor for fibronectin,are involved in adhesion of carcinoma cells to fibronectin andvitronectin. This is especially true for carcinoma cells arising fromthe malignant progression of colon cancer (Lehmann, M. et al. Cancer Res1994, 54(8), 2102-7. Furthermore, integrin expression in colon cancercells is regulated by the cytoplasmic domain of the beta-6 integrinsubunit which signals through the ERK2 pathway (Niu, J. et al. Int. J.Cancer 2002, 99(4), 529-537) and beta6 expression is associated withsecretion of gelatinase B, an enzyme involved in tumor cell invasion andmetastatic mechanisms (Agrez, et al. Int. J. Cancer 1999, 81(1), 90-97).

There is now considerable evidence that progressive tumor growth isdependent upon angiogenesis, the formation of new blood vessels, toprovide tumors with nutrients and oxygen, to carry away waste productsand to act as conduits for the metastasis of tumor cells to distantsites (Gastl et al., Oncol. 54:177-184). Recent studies have furtherdefined the roles of integrins in the angiogenic process. Duringangiogenesis, a number of integrins that are expressed on the surface ofactivated endothelial cells regulate critical adhesive interactions witha variety of ECM proteins to regulate distinct biological events such ascell migration, proliferation and differentiation. Specifically, theclosely related but distinct integrins αVβ3 and αVβ5 have been shown tomediate independent pathways in the angiogenic process. An antibodygenerated against αVβ3 blocked basic fibroblast growth factor (bFGF)induced angiogenesis, whereas an antibody specific to αVβ5 inhibitedvascular endothelial growth factor (VEGF) induced angiogenesis(Eliceiri, et al., J. Clin. Invest. 103: 1227-1230 (1999); Friedlanderet al., Science 270: 1500-1502 (1995)). Therefore, integrins andespecially the alpha V subunit containing integrins, are reasonabletherapeutic targets for diseases that involve angiogenesis such asdisease of the eye and neoplastic disease, tissue remodeling such asrestenosis, and proliferation of certain cells types particularlyepithelial and squamous cell carcinomas.

Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/ormonoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestionor fusion protein products thereof) are potential therapeutic agentsthat are being investigated in some cases to attempt to treat certaindiseases. However, such antibodies or fragments can elicit an immuneresponse when administered to humans. Such an immune response can resultin an immune complex-mediated clearance of the antibodies or fragmentsfrom the circulation, and make repeated administration unsuitable fortherapy, thereby reducing the therapeutic benefit to the patient andlimiting the readministration of the antibody or fragment. For example,repeated administration of antibodies or fragments comprising non-humanportions can lead to serum sickness and/or anaphylaxis. In order toavoid these and other problems, a number of approaches have been takento reduce the immunogenicity of such antibodies and portions thereof,including chimerization and humanization, as well known in the art.These and other approaches, however, still can result in antibodies orfragments having some immunogenicity, low affinity, low avidity, or withproblems in cell culture, scale up, production, and/or low yields. Thus,such antibodies or fragments can be less than ideally suited formanufacture or use as therapeutic proteins.

Accordingly, there is a need to provide human antibodies toanti-integrin alpha-V subunit antibodies or fragments thereof thatovercome one or more of these problems, as well as improvements overknown antibodies or fragments thereof.

SUMMARY OF THE INVENTION

The present invention provides isolated human anti-integrin alpha-Vsubunit antibodies, immunoglobulins, cleavage products and otherspecified portions and variants thereof, as well as anti-alpha-V subunitantibody compositions, encoding or complementary nucleic acids, vectors,host cells, compositions, formulations, devices, transgenic animals,transgenic plants, and methods of making and using thereof, as describedand enabled herein, in combination with what is known in the art. Theantibodies of the invention bind the various forms of the alpha Vsubunit of the integrin receptor with particular affinity andspecificity regardless of the various beta subunits of the integrinheterodimer to which the alpha V subunit is paired. Accordingly, theantibodies can be used in a variety of methods for diagnosing, treating,and/or preventing diseases involving cell adhesion mediated by theintegrin receptor, particularly diseases involving alpha V integrinmediated angiogenesis, such as prostate cancer, colon cancer, and renalcarcinoma.

Thus, in one embodiment, the present invention provides at least oneisolated anti-integrin alpha-V subunit antibody as described herein. Inone embodiment, the antibody according to the present invention includesany protein or peptide containing molecule that comprises at least aportion of a complementarity determining region (CDR) of a heavy orlight chain or a ligand binding portion thereof derived from theantibody designated CNTO 95, in combination with a heavy chain or lightchain variable region, a heavy chain or light chain constant region, aframework region, or any portion thereof, that can be incorporated intoan antibody of the present invention. The antibody CNTO 95 describedherein is a human anti-alpha V antibody derived from immunization of atransgenic mouse containing genes for the expression of humanimmunoglobulins. Thus, in one embodiment, the invention is directed toantibodies containing at least one CDR region or variable region derivedfrom the CNTO 95 antibody. An antibody of the invention can include orbe derived from any mammal, such as but not limited to a human, a mouse,a rabbit, a rat, a rodent, a primate, or any combination thereof, andthe like, or the antibody can be derived from a synthetic source, suchas a synthetic phage display library.

Particular therapeutic antibodies of the invention include humanmonoclonal antibody CNTO 95, and functionally equivalent antibodieswhich have the human heavy chain and human light chain amino acidsequences in their variable regions as set forth in SEQ ID NO: 7 and SEQID NO: 8 respectively, and conservative modifications thereof.

Still other particular human antibodies of the invention include thosewhich comprise a CDR domain having a human heavy and light chain CDR1region, a human heavy and light chain CDR2 region, and a human heavy andlight chain CDR3 region, wherein (a) the CDR1, CDR2, and CDR3 of thehuman heavy chain regions comprise an amino acid sequence selected fromthe group consisting of the amino acid sequences of the CDR1, CDR2, andCDR3 regions shown in SEQ ID NOs: 1, 2 and 3, and conservative sequencemodifications thereof, and (b) the CDRI, CDR2, and CDR3 of the humanlight chain regions comprise an amino acid sequence selected from thegroup consisting of the amino acid sequences of the CDRI, CDR2, and CDR3regions shown in SEQ ID Nos: 3, 4 and 5, and conservative sequencemodifications thereof. The antibody amino acid sequence can furtheroptionally comprise at least one specified substitution, insertion ordeletion as described herein or as known in the art.

Other particular antibodies of the invention include human monoclonalantibodies which bind to an epitope defined by antibody CNTO 95, and/orwhich compete for binding to the alpha V integrin subunit with antibodyCNTO 95, or which have other functional binding characteristicsexhibited by antibody CNTO 95. Such antibodies include, for example,those which bind to alpha V with a dissociation constant (KD) Of 10⁻⁷ Mor less, such as of 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰M or less, oreven lower (e.g., 10-″M or less). Such antibodies include those whichcompetitively inhibit binding of the CNTO 95 antibody to human alpha-Vintegrin. Such antibodies further include those which exhibit no crossreactivity with murine anti alpha V antibodies LM609, P1F6, or VNR139.

Isolated human antibodies of the invention include a variety of antibodyisotypes, such as IgG1, (e.g., IgG1k), IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgAsec, IgD, and IgE. The antibodies can be full-length antibodies(e.g., IgG1 or IgG3) or can include only an antigen-binding portion(e.g., a Fab, F(ab′) 2, Fv, or a single chain Fv fragment).

At least one antibody of the invention binds at least one specifiedepitope specific to at least one integrin alpha-V subunit protein,fragment, portion or any combination thereof. The epitope can compriseat least one antibody binding region that comprises at least one portionof said protein, which epitope is preferably comprised of at least 1-5amino acids of at least one portion of an alpha-V subunit, such as butnot limited to, (a) 29-48, 58-63, 69-79, 82-85, 88-134, 140-157,161-183, 186-190, 192-198, 202-212, 215-217, 223-237, 240-244, 248-255,259-268, 287-301, 313-322, 326-328, 332-344, 348-351, 354-365, 376-387,393-401, 407-414, 417-419, 422-433, 443-451, 458-461, 465-469, 472, (b)32-41, 46-47, 53-55, 58-69, 72-74, 77-79, 85-88, 91-94, 96-105, 110-113,117-125, 129-142, 145-153, 155-159, 161-163, 166-170, 172-174, 184-197,200-209, 215-218, 221-225, 184-197, 200-209, 215-218, 221-225, 227-250,259-261, 263-267, 269-270, 275-281; and (c) 29-35, 43-45, 48-63, 67-69,72-74, 80-82, 84-87, 95-105, 108-113, 117-142, 145-163, 166-170,172-176, 184-186, 191-201, 204-206, 216-219, 224-226, 229-251, 260-262,264-268, 276-282, 286-288, 294-299, 301-318, 323-325, 328-330, 338-342,345-349, 353-358, of SEQ ID NO: 9, 16, and 17, respectively thereof, orsuch as but not limited to, at least one functional, extracellular,soluble, hydrophilic, external or cytoplasmic domain of said alpha-Vsubunit protein, or any portion thereof. Particularly preferred areantibodies which bind to substantially the same epitope on the alpha Vintegrin subunit defined by the epitope of CNTO 95, and/or which competefor binding to alpha V integrin with antibody CNTO 95, or which haveother functional binding characteristics exhibited by antibody CNTO 95.

The present invention also provides at least one isolated anti-alpha-Vsubunit antibody as described herein, wherein the antibody has at leastone activity, such as, but not limited to inhibition of vitronectinbinding, inhibition of binding of alpha-V beta-3 to at least one of analpha-V beta3 ligand or receptor, inhibition of binding of alpha-Vbeta-5 to at least one of an alpha-V beta-5 ligand or receptor,inhibition of binding of alpha-V beta-6 to at least one of an alpha-Vbeta-6 ligand or receptor, angiogenesis modulation, binding to alpha-Vsubunit or single integrin expressing cells. A(n) anti-alpha-V subunitantibody can thus be screened for a corresponding activity according toknown methods, such as but not limited to, competition with the CNTO 95antibody for at least one biological activity towards an integrinalpha-V subunit protein.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding the specific anti-integrin alpha-V subunit antibodies describedherein. Such nucleic acid molecules include those encoding all or aportion of a human monoclonal anti-alpha V antibody as described herein(e.g., which encode at least one light or heavy chain CDR of theantibody), as well as recombinant expression vectors which include suchnucleic acids, and host cells transfected with such vectors. Methods ofproducing the antibodies by culturing such host cells are alsoencompassed by the invention. Particular nucleic acids provided by theinvention comprise the nucleotide sequences shown in SEQ ID NOs: 10, 11and 12 and SEQ ID NOs: 13, 14 and 15, which encode the heavy and lightchains CDRs, respectively, of human anti-alpha V antibody CNTO 95 andthe nucleic acids which encode the complete variable region of the heavyor light chain, respectively, as shown in SEQ ID Nos: 18 and 19. Thepresent invention further provides recombinant vectors comprising saidanti-integrin alpha-V subunit antibody nucleic acid molecules, hostcells containing such nucleic acids and/or recombinant vectors, as wellas methods of making and/or using such antibody nucleic acids, vectorsand/or host cells.

The present invention also provides at least one method for expressingat least one anti-alpha-V subunit antibody as described herein, oralpha-V subunit anti-idiotype antibody as described herein, in a hostcell, comprising culturing a host cell as described herein underconditions wherein at least one anti-alpha-V subunit antibody isexpressed in detectable and/or recoverable amounts.

The present invention also provides at least one composition comprising(a) an isolated anti-alpha-V subunit antibody encoding nucleic acidand/or antibody as described herein; and (b) a suitable carrier ordiluent. The carrier or diluent can optionally be pharmaceuticallyacceptable, according to known carriers or diluents. The composition canoptionally further comprise at least one further compound, protein orcomposition.

The present invention further provides at least one anti-alpha-V subunitantibody method or composition, for administering a therapeuticallyeffective amount to modulate or treat at least one alpha-V subunitrelated condition in a cell, tissue, organ, animal or patient and/or,prior to, subsequent to, or during a related condition, as known in theart and/or as described herein. The compositions include, for example,pharmaceutical and diagnostic compositions/kits, comprising apharmaceutically acceptable carrier and at least one human anti-alpha Vantibody, or an antigen-binding portion thereof. In one embodiment, thecomposition comprises a combination of human antibodies orantigen-binding portions thereof, preferably each of which binds to adistinct epitope. For example, a pharmaceutical composition comprising ahuman monoclonal antibody that mediates highly effective killing oftarget cells in the presence of effector cells can be combined with thehuman monoclonal antibody hereof that inhibits the growth of cellsexpressing alpha V integrin. Thus, the combination provides multipletherapies tailored to provide the maximum therapeutic benefit.Compositions, e.g., pharmaceutical compositions, comprising acombination of at least one human anti alpha V-antibody, orantigen-binding portion thereof, and at least one bispecific ormultispecific molecule of the invention, are also within the scope ofthe invention.

In yet another aspect of the invention, the human anti-alpha Vantibodies are derivatized, linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein (e.g., an Fab′fragment). For example, an antibody or antigen-binding portion of theinvention can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., to produce abispecific or a multispecific antibody), a cytotoxin, a cellular ligandor an antigen. Accordingly, present invention encompasses a largevariety of antibody conjugates, bi- and multispecific molecules, andfusion proteins, all of which bind to alpha V expressing cells and whichtarget other molecules to the cells, or which bind to alpha V and toother molecules or cells.

Alternatively, human antibodies of the invention can be co-administeredwith such therapeutic and cytotoxic agents, but not linked to them. Theycan be coadministered simultaneously with such agents (e.g., in a singlecomposition or separately) or can be administered before or afteradministration of such agents. Such agents can include chemotherapeuticagents, such as dacarbazine, doxorubicin (adriamycin), cisplatin,bleomycin sulfate, carmustine, chlorambucil, cyclophosphamidehydroxyurea and combinations thereof. Human antibodies of the inventionalso can be administered in conjunction with radiation therapy.

In yet another embodiment, the present invention provides a method forinhibiting the proliferation and/or growth of a cell expressing alpha Vintegrin, and/or inducing killing of a cell expressing alpha V integrin,by contacting the cells with (e.g., administering to a subject) one ormore human antibodies of the invention and/or related therapeuticcompositions, derivatives etc. containing the antibodies as describedabove. In a particular embodiment, the method comprises contacting cellsexpressing alpha V integrin either in vitro or in vivo with one or acombination of human anti-alpha V antibodies of the invention in thepresence of a human effector cell. The method can be employed inculture, e.g. in vitro or ex vivo (e.g., cultures comprising cellsexpressing alpha V and effector cells). For example, a sample containingcells expressing alpha V and effector cells can be cultured in vitro,and combined with an antibody of the invention.

Alternatively, the method can be performed in a subject, e.g., as partof an in vivo (e.g., therapeutic or prophylactic) protocol. For use inin vivo treatment and prevention of alpha V mediated diseases, humanantibodies of the present invention are administered to patients (e.g.,human subjects) at therapeutically effective dosages (e.g., to inhibit,eliminate or prevent growth of cells expressing alpha V or to inhibitangiogenesis and thus inhibit the growth of cells where growth ismediated by angiogenesis) using any suitable route of administration forantibody-based clinical products as are well known in the art, such asby injection or infusion.

Accordingly, human antibodies of the present invention can be used totreat and/or prevent a variety of alpha V integrin mediated diseases byadministering a suitable dosage (or series of dosages) of the antibodiesto patients suffering from such diseases. Exemplary diseases that can betreated (e.g., ameliorated) or prevented using the methods andcompositions of the invention include, but are not limited to, cancers,such as metastatic melanoma, prostate cancer, colon cancer, and renalcarcinoma.

In a particular embodiment of the invention, the patient can beadditionally treated with a chemotherapeutic agent, radiation, or anagent that modulates, e.g., enhances, the expression or activity of anFc receptor, such as a cytokine. Typical cytokines for administrationduring treatment include granulocyte colony-stimulating fact or (G-CSF),granulocytemacrophage colony-stimulating factor (GM-CSF), interferon-γ(IFN-γ), and tumor necrosis factor (TNF). Typical therapeutic agentsinclude, among others, anti-neoplastic agents such as dacarbazine,doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine,chlorambucil, cyclophosphamide, and hydroxyurea.

In yet another aspect, the present invention provides a transgenicnonhuman animal, such as a transgenic mouse (also referred to herein asa “HuMAb mouse”), which expresses a fully human monoclonal antibody thatbinds to alpha V. In a particular embodiment, the transgenic nonhumananimal is a transgenic mouse having a genome comprising a human heavychain transgene and a human light chain transgene encoding all or aportion of an anti-alpha V antibody of the invention. To generate humananti-alpha V antibodies, the transgenic nonhuman animal can be immunizedwith a purified or enriched preparation of alpha V antigen and/or cellsexpressing alpha V. Preferably, the transgenic nonhuman animal, e.g.,the transgenic mouse, is capable of producing multiple isotypes of humanmonoclonal antibodies to Alpha V (e.g., IgG, IgA and/or IgM) byundergoing V-D-J recombination and isotype switching. Isotype switchingmay occur by, e.g., classical or non-classical isotype switching.

Accordingly, in another embodiment, the invention provides isolatedcells derived from a transgenic nonhuman animal as described above,e.g., a transgenic mouse, which express human anti-alpha V antibodies.The isolated B-cells can then be immortalized by fusion to animmortalized cell to provide a source (e.g., a hybridoma) of humananti-alpha V antibodies. Such hybridomas (i.e., which produce humananti-ALPHA V antibodies) are also included within the scope of theinvention.

As exemplified herein, human anti-alpha V antibodies can be obtaineddirectly from hybridomas which express the antibody, or can be clonedand recombinantly expressed in a host cell, such as a transfectoma(e.g., a transfectoma consisting of immortalized CHO cells orlymphocytic cells). Accordingly, the present invention provides methodsfor producing human monoclonal antibodies which bind to human alpha V.In a particular embodiment, the method includes immunizing a transgenicnonhuman animal, e.g., a transgenic mouse, as previously described(e.g., having a genome comprising a human heavy chain transgene and ahuman light chain transgene encoding all or a portion of an anti-alpha Vantibody), with a purified or enriched preparation of human alpha Vantigen and/or cells expressing human alpha V. B cells (e.g., splenic Bcells) of the animal are then obtained and fused with myeloma cells toform immortal, hybridoma cells that secrete human monoclonal antibodiesagainst alpha V.

The present invention further provides at least one anti-alpha-V subunitantibody method or composition, for diagnosing at least one alpha-Vsubunit related condition in a cell, tissue, organ, animal or patientand/or, prior to, subsequent to, or during a related condition, as knownin the art and/or as described herein.

The present invention further provides at least one alpha-V subunitanti-idiotype antibody to at least one alpha-V subunit antibody of thepresent invention. The anti-idiotype antibody includes any protein orpeptide containing molecule that comprises at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into anantibody of the present invention. An anti-idiotype antibody of theinvention can include or be derived from any mammal, such as but notlimited to a human, a mouse, a rabbit, a rat, a rodent, a primate, andthe like.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding at least one alpha-V subunit anti-idiotype antibody, comprisingat least one specified sequence, domain, portion or variant thereof. Thepresent invention further provides recombinant vectors comprising saidalpha-V subunit anti-idiotype antibody encoding nucleic acid molecules,host cells containing such nucleic acids and/or recombinant vectors, aswell as methods of making and/or using such anti-idiotype antibodynucleic acids, vectors and/or host cells.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of doubling dilutions of anti-αVβ3 Mabs which wereincubated on αVβ3 coated plates for 1 hour at RT. Plates were washedtwice and probed with HRP labeled goat anti-human IgG kappa specificantibody for 1 hour at RT. Plates were again washed, developed with OPDsubstrate and OD's measured at 490 nm.

FIG. 2 shows a graph of calcein-labeled M21 cells which werepreincubated with antibody samples in the absence or presence of P1F6,anti-αVβ5 ascites for 30 minutes, then added to vitronectin coatedplates for 45 minutes. Non-bound M21 cells were removed with two 150μL/well washes with HBSS with calcium. Plate was read on a fluorometerat 485-538 nm.

FIG. 3 shows a graph of cell adhesion where MDA MB 435L2 cells wereharvested and pre-incubated with various concentrations of CNTO95 for 10minutes. Tumor cells were then added to vitronectin coated Linbro platesand incubated at 37° C. for one hour. Wells were washed three times andthe MTT based Cell Titer AQ dye was added to each well. Cell adhesionwas determined in an ELISA plate reader where OD490 nm is directlyproportional to cell adhesion. Cell adhesion to BSA coated wells servedas negative control (data not shown). Each data point is the mean oftriplicate determinations.

FIGS. 4A-D show graphs of antibody binding to αvβ3 where this ligand waspreincubated in doubling dilutions starting at 10 ug/mL with 50 mM EDTAin 1% BSA-HBSS (in the absence of Ca++) or with 1% BSA-HBSS (with Ca++)for 30 min, 37EC. Mixtures added to plates coated with CNTO 95, C372,c7E3 or LM609 IgG and incubated for 1 hour, 37° C. LM609 or CNTO 95added at 20: g/mL in appropriate buffer (+/−Ca++) for 30 min, 37° C.Plates probed with goat anti-mouse IgG Fc, HRP or goat anti-human IgGFc, HRP.

FIGS. 4E-G show graphs of antibody binding to a alphaVbeta5, where thisligand was preincubated in doubling dilutions starting at 10 ug/mL with50 mM EDTA in 1% BSA-HBSS (in the absence of Ca++) or with 1% BSA-HBSS(with Ca++) for 30 min, 37° C. Mixtures added to plates coated with CNTO95, C372, c7E3 IgG and incubated for 1 hour, 37° C. VNR139 was added at10 mg/mL in appropriate buffer (+/−Ca++) for 30 min, 37° C. Platesprobed with goat anti-mouse IgG Fc, HRP.

FIG. 5A-B shows a graph of saturation binding curve of CNTO 95 (FIG. 5A)and abciximab (an anti-gpIIb/IIIa/αvB3 antibody) (FIG. 5B) on αvβ3coated plates.

FIG. 6A-B shows a graph of saturation binding curve of CNTO 95 (FIG. 5A)and abciximab (FIG. 5B) on αvβ5 coated plates.

FIG. 7A-C shows saturation binding curves with graphs binding to (FIG.7A): A375S2; (FIG. 7B): HT-29; (FIG. 7C): M21. Cells were plated 2 daysprior to experiment, and 1×10⁵ cells/well at the time of study. 125-ICNTO 95 (1 μCi/μg) was added in 1% growth media and incubated on cellsfor 1.5 h, 37° C. Nonspecific binding was determined using 100× cold mAbin media. Cells were washed 3× and counted for bound radioactivity. Eachcurve represents 4-5 separate studies, and each data point in anexperiment was the mean of triplicate samples.

FIG. 8A-C shows saturation binding curves with graphs binding to (FIG.8A): A375S2; (FIG. 8B): HT-29; (FIG. 8C): M21. Cells were plated 2 daysprior to the experiment, and 1×10⁵ cells/well at the time of study.125-I abciximab (1 μCi/μg) was added in 1% growth media and incubated oncells for 1.5 h, 37° C. Nonspecific binding was determined using 100×cold mAb in media. Cells were washed 3× and counted for boundradioactivity. Each curve represents 4-5 separate studies, and each datapoint in an experiment was the mean of triplicate samples.

FIG. 9 shows a representation of microcapillary formation of endothelialcells from MC beads cultured in fibrin gels. Objective lens: 40×. Theassay was performed as described in Methods of Example 4.

FIG. 10 shows a graph of quantification of capillary formation in afibrin gel in media containing 30 ng/ml bFGF dissolved in 0.1% serum.The number of microcapillary sprouts were quantified as described inMethods of Example 4. Control indicates vehicle control, mouse (M) andhuman (H) IgG served as negative controls. LM-P1F6 is a combination ofboth LM609 and P1F6. Each bar represents the mean of 6 wells (+/−SD).

FIG. 11 shows a graph of quantification of capillary formation in afibrin gel in complete media. The number of microcapillary sprouts werequantified as described in Methods of Example 4. Control indicatesvehicle control. Mouse (m) and human (h)-IgG served as negativecontrols. LM-P1F6 is a combination of both LM609 and P1F6. Each barrepresents the mean of 6 wells (+/−SD).

FIG. 12. HT29 cells (FIGS. 12A, B and C) express αvβ5, but not αvβ3integrin on their surface. HUVEC (FIGS. 12D, E and F) and A375S.2 (FIGS.12G, H and I) cells express αvβ5 and αvβ3 integrin on their surface.Tumor cells and endothelial cells were stained by immunofluorescence andanalyzed by flow cytometry. The histogram on the left representsbackground fluorescence in the presence of isotype matched antibody. Thehistogram on the right indicates positive staining. A, D, G, LM609 (mAbdirected to αvβ3, 10 μg/ml); B, E, H, PIF6 (mAb directed to αvβ5, 10μg/ml); and C, F, I, GenO95 (10 μg/ml).

FIG. 13. Adhesion of HUVECS to matrix protein-coated plates. Adhesionassay was performed as described in Methods of Example 5. Plate was readon a fluorometer at 485-538 nm. Cell adhesion to BSA coated wells servedas a negative control. In FIG. 13, the extent of cell adhesion in thepresence of various concentrations of antibody was plotted as a percentof cell adhesion in the absence of antibody that was considered as 100%.Each data point is the mean of triplicate determinations (+/−SD).

FIG. 14. Adhesion of human melanoma cells to matrix protein-coatedplates. Adhesion assay was performed as described in Methods of Example5. Cell adhesion to BSA coated wells served as a negative control. InFIG. 14 the extent of cell adhesion in the presence of variousconcentrations of antibody was plotted as a percent of cell adhesion inthe absence of antibody that was considered as 100%. Each data point isthe mean of triplicate determinations (+/−SD).

FIG. 15. Adhesion of human colon carcinoma HT29 cells to vitronectin.The adhesion assay was performed as described in the Examples. Celladhesion to BSA coated wells served as a negative control. Data in FIG.15 are plotted as percent of maximum binding (absence of antibody), andare the mean of triplicate determinations (+/−SD).

FIG. 16A-D. Migration of HUVECS toward 2 μg/ml vitronectin. The assaywas performed as described in Methods and cells were allowed to migratefor 6 h. Photomicrographs are representative fields (10× objective lens)of cell migration in FIG. 16A, absence of antibody, (16B), CNTO 95 (5μg/ml), (16C), CNTO 95 (40 μg/ml). FIG. 16D is graphical representationof cell migration in the presence of varying concentrations of GenO95.The data were normalized to percent of control (no antibody) which wasconsidered as 100%, and each point is the mean of three transwellfilters (+/−SD).

FIG. 17. Migration of HUVECS toward 2 μg/ml vitronectin in the presenceof antibodies to αvβ3 and αvβ5. The migration assay was performed asdescribed in Methods of Example 5, and cells were allowed to migrate for6 hours. LM609 and P1F6 are mAbs directed to αvβ3 and αvβ5,respectively. The data shown in FIG. 17 were normalized to percent ofcontrol (no antibody) which was considered as 100%, and each bar is themean of three transwell filters (+/−SD). BSA, mouse IgG and human IgGserved as negative controls. LM609-PIF6 represents combinations of bothantibodies. The antibodies and BSA were used at a concentration of 10μg/ml.

FIG. 18A-E. Migration of HUVECS towards 2% FBS. Migration assay wasallowed to proceed for 4 h and the data was captured as described in theMethods of Example 5. FIG. 18(A) is a graphical representation of cellmigration in the presence of LM609, P1F6, combination of LM609+P1F6,isotype matched control antibodies (human and mouse). The antibodies andproteins were used at a concentration of 10 μg/ml. FIG. 18(B) is agraphical representation of cell migration in the presence of ReoPro andGenO95. Photomicrographs are representative fields (10× objective lens)of cell migration in FIG. 18(C), the absence of antibody, FIG. 18(D),GenO95 (5 μg/ml), and FIG. 18(E), GenO95 (20 μg/ml). The data werenormalized to percent of control (no antibody) which was considered as100%, and each point is the mean of three transwell filters (+/−SD).

FIG. 19A-E. Migration of A375S.2 cells toward 10% FBS. Migration assaywas allowed to proceed for 4 h and the data was captured as described inthe Methods of Example 5. Antibodies were used at a concentration of 10μg/ml. FIG. 19(A) is a graphical representation of cell migration in thepresence of varying concentrations of GenO95. FIG. 19(B) is a graphicalrepresentation of cell migration in the presence of LM609, P1F6,combination of LM609+P1F6, isotype matched control antibodies (human andmouse). The data were normalized to percent of control, which wasconsidered as 100%, and each point is the mean of three transwellfilters (+/−SD). Photomicro-graphs are representative fields (10×objective lens) of cell migration in FIG. 19(C), absence of antibody,FIG. 19(D), GenO95 (5 μg/ml), and FIG. 19(E), GenO95 (20 μg/ml).

FIG. 20A-E. Migration of HUVECS towards vitronectin in the presence ofbFGF. The undersides of migration chamber filters were coated with 2μg/ml vitronectin, and the assay was performed as described in theMethods of Example 5. Cells were allowed to migrate for 6 h. In FIG.20A-E, each data point is the mean of 3 transwell filters (+/−SD). FIG.20(A), bFGF; FIG. 20(B), CNTO 95 (5 μg/ml); FIG. 20(C), CNTO 95 (40μg/ml); FIG. 20(D), no-bFGF. FIG. 20(E), Inhibition of cell migration inthe presence of various antibodies is shown graphically.

FIG. 21A-D. Invasion of A375S.2 cells through a fibrin gel (5 mg/ml).Invasion assay was allowed to proceed for 24 h and data was captured asdescribed in the Methods of Example 5. Photomicrographs arerepresentative fields (4× objective lens) of cell invasion in FIG. 21(A)the absence of antibodies, FIG. 21(B) CNTO 95 (10 μg/ml), FIGS. 21(C)and (D) are graphical representation of cell invasion in presence ofCNTO 95, 10E5 F(ab′)₂, LM609, P1F6, LM-PIF6 (LM609+P1F6), human andmouse IgGs (H-IgG and M-IgG). Graph FIG. 21(D): The concentration of allantibodies and proteins is 10 μg/ml. The data were normalized to percentof control (no antibody) which was considered as 100%, and each point isthe mean of three transwell filters (+/−SD).

FIG. 22A-D. are histograms from flow cytometric analysis of HEK cellstransfected with various integrin DNA and immunoflurescently stained asnoted. FIG. 22(A): cells stained with antibodies for specific subunits.FIG. 22(B): mock transfected and avb6 transfected cells were analyzedfor expression of αvβ3, αvβ5, and β1 integrins. FIG. 22(C): HEK 293cells were transfected with αV, β6, or αvβ6 cDNA and CNTO 95 bindingmeasured. The vertical line serves as a reference marker and indicatesthe fluorescence intensity at which mock transfectants were <2%positive. FIG. 22(D): analysis of mock transfected (A, B, C) or αvβ6transfected (C, D, E) HEK 293 cells for CNTO 95 immunoreactivity. Cellswere stained with anti-avb6 (A, D) or CNTO 95 (B, E). Double stainingwas used to detect cells which were immunoreactive with both antibodiessimultaneously (C, F). The upper-right quadrant in F indicates thatcells which stained intensely for avb6 also stained intensely for CNTO95.

FIG. 23 is a graph showing the number of microvessels sprouting from rataortic rings treated as described. BSA (20 ug/ml) and irrelevant humanIgG (20 ug/ml) were used as negative controls. Data points represent onerat aorta, with mean values for each group indicated by lines. P valueswere determined by comparing the antibody-treated groups with theBSA-treated group using a two-tailed unpaired t-test.

FIG. 24 are graphs of the data on length FIG. 24(A) and number FIG.24(B) of blood vessels found in bFGF-impregnated Matrigel plugs in nuderats examined on day 7. Vessel number and length were assessed bymicroscopy (2×) aided by image analysis software (Phase 3 Image System).Each point represents average per view from one Matrigel sample (2plugs/animal), the line represents the group mean. A two-tailed unpairedt-test indicated P<0.0001 for all four CNTO 95 dose groups compared tothe control IgG group.

FIG. 25A-C are graphs of the data on length Figure (A), number Figure(B) and density FIG. 25(C) of blood vessels found in bFGF-impregnatedMatrigel plugs in monkeys examined on day 7. For FIG. 25(A) and FIG.25(B) each point represents average per 2× field from one Matrigelsample (4 Matrigel plugs/animal). Horizontal lines indicate mean.Matrigel alone, bFGF-PBS, 5 ug/ml bFGF. bFGF-control IgG, 5 ug/ml bFGF,10 ug/kg control IgG i.v. bFGF-CNTO 95, 5 ug/ml bFGF, 10 mg/kg CNTO 95i.v. A two-tailed unpaired t-test analysis indicated P<0.001 forMatrigel alone and bFGF-CNTO 95 groups compared to the bFGF-PBS andbFGF-control IgG groups. There was no difference between bFGF-CNTO 95and Matrigel alone groups (P>0.05). For FIG. 25(C), the percentage ofMatrigel cross-sectional area occupied by vessels was calculated usingcomputer-assisted image analysis. Each point represents the averagedensity per Matrigel sample (4 samples/animal). A two-tailed unpairedt-test analysis indicated P<0.01 for Matrigel alone and bFGF-CNTO 95groups compared to the bFGF-HBSS and bFGF-control IgG groups. There wasno difference between bFGF-CNTO 95 and Matrigel alone groups (P>0.05).

FIGS. 26A and B are graphs of the data on length FIG. 26(A) and numberFIG. 26(B) of blood vessels found in bFGF-impregnated Matrigel plugs innude rats. Each point represents average per view from one Matrigel plugand the line is the mean from 10 plugs (2 plugs/per animal). Insiderefers to CNTO 95 (40 mg/ml) that was included in the Matrigel solutionprior to injection. IV refers to CNTO 95 (10 mg/kg) that was injectedintravenously as a bolus after the Matrigel solution was injected intothe rats. Inside +IV refers to CNTO 95 that was mixed with the Matrigelsolution and injected intravenously. A two-tailed unpaired t-testanalysis indicated P<0.001 for all three CNTO 95 groups compared to thecontrol IgG group. The three CNTO 95 groups were not statisticallydifferent from each other.

FIG. 27. is a graph showing the change volume over time of a humanmelanoma tumor in nude mice and the effect of administering CNTO 95.Mice were inoculated subcutaneously with A375.S2 cells (3×106), anddosing with CNTO 95 or control was initiated three days later. Mice weretreated with CNTO 95 or vehicle three times per week at a dose of 10mg/kg i.p. Each data point is the mean tumor volume from 10tumor-bearing animals (+SEM). CNTO 95 given three times per weeksignificantly inhibited growth of tumors when compared to controltreated animals at day 26 (P=0.0005).

FIG. 28. is a graph showing the change volume over time of a humanmelanoma tumor in nude rats and the effect of administering CNTO 95.Rats were inoculated subcutaneously with A375.S2 cells (3×106), andtherapy with CNTO 95 or control was initiated three days later. Ratswere treated with CNTO 95 or vehicle once per week at a dose of 10 mg/kgi.v. Each data point is the mean tumor volume from 9 tumor-bearinganimals (+SEM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticanti-alpha-V subunit human monoclonal antibodies and alpha-V subunitanti-idiotype antibodies thereto, as well as compositions and encodingnucleic acid molecules comprising at least one polynucleotide encodingat least one anti-alpha-V subunit antibody or anti-idiotype antibody.The present invention further includes, but is not limited to, methodsof making and using such nucleic acids and antibodies and anti-idiotypeantibodies, including diagnostic and therapeutic compositions, methodsand devices. Therapies of the invention employ isolated human monoclonalantibodies and/or related compositions containing the antibodies whichbind to an epitope present on the alpha V integrin subunit and iscapable of blocking the binding of various alpha V containing integrins,regardless of the beta subunit to which it is associated. In aparticular embodiment exemplified herein, the human antibodies areproduced in a nonhuman transgenic animal, e.g., a transgenic mouse.Accordingly, aspects of the invention include not only antibodies,antibody fragments, and pharmaceutical compositions thereof, but alsononhuman transgenic animals, B-cells and hybridomas which producemonoclonal antibodies. Methods of using the antibodies of the inventionto detect a cell expressing alpha V, or to inhibit growth,differentiation and/or motility of a cell expressing alpha V, either invitro or in vivo, are also encompassed by the invention.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “alpha V integrin”, “alpha V subunit integrin”, and “alpha Vsubunit containing integrin” are used interchangeably herein to meanAlpha V transmembrane glycoprotein subunits of a functional integrinsheterodimer and include all of the variants, isoforms and specieshomologs of alpha V. Accordingly, human antibodies of the invention may,in certain cases, cross-react with alpha V from species other thanhuman, or other proteins which are structurally related to human alpha V(e.g., human alpha V homologs). In other cases, the antibodies may becompletely specific for human alpha V and not exhibit species or othertypes of cross-reactivity.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the antibody includesany protein or peptide containing molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, which can be incorporated into an antibody of thepresent invention. An “alpha V antibody”, “alpha V subunit antibody” or“alpha V integrin antibody” is an antibody that affects the alpha Vligand, such as but not limited to where such antibody modulates,decreases, increases, antagonizes, angonizes, mitigates, alleviates,blocks, inhibits, abrogates and/or interferes with at least one alpha-Vsubunit activity or binding, or with alpha-V subunit receptor activityor binding, in vitro, in situ and/or in vivo. As a non-limiting example,a suitable anti-alpha-V subunit antibody, specified portion or variantof the present invention can bind at least one alpha-V subunit, orspecified portions, variants or domains thereof. A suitable anti-alpha-Vsubunit antibody, specified portion, or variant can also optionallyaffect at least one of alpha-V subunit activity or function, such as butnot limited to, RNA, DNA or protein synthesis, alpha-V subunit release,alpha-V subunit receptor signaling, membrane alpha-V subunit cleavage,alpha-V subunit activity, alpha-V subunit production and/or synthesis.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, includingantibody mimetics or comprising portions of antibodies that mimic thestructure and/or function of an antibody or specified fragment orportion thereof, including single chain antibodies and fragmentsthereof. Functional fragments include antigen-binding fragments thatbind to a mammalian alpha-V subunit. Examples of binding fragmentsencompassed within the term “antigen binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH, domains; (ii) a F(ab′) 2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH, domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (I 988) Science 242:423-426, and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The term “native conformational epitope” or “native proteinepitope” are used interchangeably herein, and include protein epitopesresulting from conformational folding of the integrin molecule whicharise when amino acids from differing portions of the linear sequence ofthe integrin molecule come together in close proximity in 3 dimensionalspace. Such conformational epitopes are distributed on the extracellularside of the plasma membrane.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with, (a) a cell surface antigen and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g. aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with, (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to cell surface antigens, such as alpha V, and to othertargets, such as Fc receptors on effector cells.

The term “bispecific antibodies” also includes diabodies. Diabodies arebivalent, bispecific antibodies in which the VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (I 994) Structure 2:1121-1123). Bispecific,heterospecific, heteroconjugate or similar antibodies can also be usedthat are monoclonal, preferably human or humanized, antibodies that havebinding specificities for at least two different antigens. In thepresent case, one of the binding specificities is for at least onealpha-V subunit protein, the other one is for any other antigen. Methodsfor making bispecific antibodies are known in the art. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

As used herein, the term “heteroantibodies” refers to two or moreantibodies, antibody binding fragments (e.g., Fab), derivativestherefrom, or antigen binding regions linked together, at least two ofwhich have different specificities. These different specificitiesinclude a binding specificity for an Fc receptor on an effector cell,and a binding specificity for an antigen or epitope on a target cell,e.g., a tumor cell.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H)1, C_(H)2,C_(H)3), hinge, (V_(L), V_(H))) is substantially non-immunogenic inhumans, with only minor sequence changes or variations. Similarly,antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent(mouse, rat, rabbit, guinea pig, hamster, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. Further, chimeric antibodies include anycombination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody. It is pointed out that ahuman antibody can be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it cancomprise a linker peptide that is not found in native human antibodies.For example, an Fv can comprise a linker peptide, such as two to abouteight glycine or other amino acid residues, which connects the variableregion of the heavy chain and the variable region of the light chain.Such linker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the gemiline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences.

In one embodiment, the human monoclonal antibodies are produced by ahybridoma which includes a B cell obtained from a transgenic nonhumananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a light chain transgene fused to animmortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further in Section I,below), (b) antibodies isolated from a host cell transformed to expressthe antibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regionsderived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies can be subjectedto in vitro mutagenesis (or, when an animal I 0 transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to Alpha V is substantially free of antibodies thatspecifically bind antigens other than Alpha V). An isolated antibodythat specifically binds to an epitope, isoform or variant of human AlphaV may, however, have cross-reactivity to other related antigens, e.g.,from other species (e.g., Alpha V species homologs). Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals. In one embodiment of the invention, a combination of“isolated” monoclonal antibodies having different specificities arecombined in a well defined composition.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10-7 M or less, and binds to the predeterminedantigen with a K_(D) that is at least twofold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) Of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to an antibody having aK_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M or less. The term“Kassoc” or “Ka”, as used herein, is intended to refer to theassociation rate of a particular antibody-antigen interaction, whereasthe term “Kdis” or “Kd,” as used herein, is intended to refer to thedissociation rate of a particular antibody-antigen interaction, The term“K_(D)”, as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) andis expressed as a molar concentration (M).

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA. The term “isolated nucleic acid molecule,” as used herein inreference to nucleic acids encoding antibodies or antibody portions(e.g., VH, VL, CDR3) that bind to Alpha V, is intended to refer to anucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thanAlpha V, which other sequences may naturally flank the nucleic acid inhuman genomic DNA. In one embodiment, the human anti-Alpha V antibody,or portion thereof, includes the nucleotide or amino acid sequence ofCNTO 95, as well as heavy chain (VH) and light chain (VL) variableregions having the amino acid sequences shown in SEQ ID NOs: 7 and 8,respectively, and nucleotide sequences encoding them, including SEQ IDNos: 18 and 19.

As disclosed and claimed herein, the sequences set forth in SEQ ID NOs.1-8 and 10-15 include “conservative sequence modifications”, i.e.,nucleotide and amino acid sequence modifications which do notsignificantly affect or alter the binding characteristics of theantibody encoded by the nucleotide sequence or containing the amino acidsequence. Such conservative sequence modifications include nucleotideand amino acid substitutions, additions and deletions. Modifications canbe introduced into SEQ ID NOs: 1-8 and 10-15 by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions include ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-Alpha Vantibody is preferably replaced with another amino acid residue from thesame side chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-Alpha V antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-AlphaV antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the (heavy and light chain variableregion) nucleotide sequences disclosed herein and/or containing the(heavy and light chain variable region) amino acid sequences disclosedherein (i.e., SEQ ID NOs: 1-8) include substantially similar antibodiesencoded by or containing similar sequences which have beenconservatively modified. Further discussion as to how such substantiallysimilar antibodies can be generated based on the partial (i.e., heavyand light chain variable regions) sequences disclosed herein as SEQ IDNOs: 1-8 is provided below. For nucleic acids, the term “substantialhomology” indicates that two nucleic acids, or designated sequencesthereof, when optimally aligned and compared, are identical, withappropriate nucleotide insertions or deletions, in at least about 80% ofthe nucleotides, usually at least about 90% to 95%, and more preferablyat least about 98% to 99.5% of the nucleotides. Alternatively,substantial homology exists when the sequences hybridize under selectivehybridization conditions, to the complement of segments with the strand.The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available at http:Hwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percentidentity between two nucleotide or amino acid sequences can alsodetermined using the algorithm of E. Meyers and W. Miller (Comput. AppLBiosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM 1 20 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm whichhas been incorporated into the GAP program in the GCG software package(available at http://www.gcg.com), using either a Blossurn 62 matrix ora PAM2 5 0 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. The nucleic acid and proteinsequences of the present invention can further be used as a “querysequence” to perform a search against public databases to, for example,identify related sequences. Such searches can be performed using theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. (I 990) J.Mol. Biol. 215.403-1 0. BLAST nucleotide searches can be performed withthe NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to the nucleic acid molecules of the invention.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences homologous to theprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25(17):3389 When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. See http:Hwww.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof, may be mutated inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, CHO cells andlymphocytic cells.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and nonmammals, such as nonhuman primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

The terms “transgenic, nonhuman animal” refers to a nonhuman animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces humananti-Alpha V antibodies when immunized with alpha V antigen and/or cellsexpressing Alpha V. The human heavy chain transgene can be integratedinto the chromosomal DNA of the mouse, as is the case for transgenic,e.g., HuMAb mice, or the human heavy chain transgene can be maintainedextrachromosomally, as is the case for transchromosomal (e.g., KM) miceas described in WO 02/43478. Such transgenic and transchromosomal miceare capable of producing multiple isotypes of human monoclonalantibodies to Alpha V (e.g., IgG, IgA and/or IgE) by undergoing V-D-Jrecombination and isotype switching.

Anti-alpha-V subunit antibodies (also termed alpha-V subunit antibodies)useful in the methods and compositions of the present invention arecharacterized by high affinity binding to alpha-V subunit and preferablyhaving low toxicity. In particular, an antibody, specified fragment orvariant of the invention, where the individual components, such as thevariable region, constant region and framework, individually and/orcollectively, optionally and preferably possess low immunogenicity, areuseful in the present invention. The antibodies that can be used in theinvention are optionally characterized by their ability to treatpatients for extended periods with measurable alleviation of symptomsand low and/or acceptable toxicity. Low or acceptable immunogenicityand/or high affinity, as well as other suitable properties, cancontribute to the therapeutic results achieved. “Low immunogenicity” isdefined herein as raising significant HAHA, HACA or HAMA responses inless than about 75%, or preferably less than about 50% of the patientstreated and/or raising low titres in the patient treated (less thanabout 300, preferably less than about 100 measured with a double antigenenzyme immunoassay) (Elliott et al., Lancet 344:1125-1127 (1994),entirely incorporated herein by reference).

Citations

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. The followingreferences are entirely incorporated herein by reference: Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

1. Production of Antibodies

Anti-alpha-V subunit antibodies of the present invention can beoptionally produced by a variety of techniques, including conventionalmonoclonal antibody techniques, e.g., the standard somatic cellhybridization technique of Kohler and Milstein (1975) Nature 256:495. Avariety of cell lines, mixed cell lines, an immortalized cell or clonalpopulation of immortalized cells, can be used, as well known in the art.See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology,John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y. (1989); Harlow and Lane, antibodies, a Laboratory Manual,Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

Human antibodies that are specific for human alpha-V subunit proteins orfragments thereof can be raised against an appropriate immunogenicantigen, such as isolated and/or alpha-V subunit protein or a portionthereof (including synthetic molecules, such as synthetic peptides).Other specific or general mammalian antibodies can be similarly raised.Preparation of Immunogenic Antigens, and Monoclonal Antibody Productioncan be Performed using any suitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO,PerC.6, YB2/O or the like, or heteromyelomas, fusion products thereof,or any cell or fusion cell derived therefrom, or any other suitable cellline as known in the art. See, e.g., www.atcc.org, www.lifetech.com.,and the like, with antibody producing cells, such as, but not limitedto, isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference. Antibody producing cells canalso be obtained from the peripheral blood or, preferably the spleen orlymph nodes, of humans or other suitable animals that have beenimmunized with the antigen of interest. Any other suitable host cell canalso be used for expressing heterologous or endogenous nucleic acidencoding an antibody, specified fragment or variant thereof, of thepresent invention. The fused cells (hybridomas) or recombinant cells canbe isolated using selective culture conditions or other suitable knownmethods, and cloned by limiting dilution or cell sorting, or other knownmethods. Cells which produce antibodies with the desired specificity canbe selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and applications)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V.,Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/research_tools.html;www.mgen.uniheidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/˜mrc7/mikeimages.html; www.antibodyresource.com/;mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;pathbox.wustl.edu/˜hcenter/index.html; www.biotech.ufl.edu/˜hcl/;

www.pebio.com/pa/340913/340913.html;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com/table.asp;

www.icnet.uk/axp/facs/davies/links.html;www.biotech.ufl.edu/˜fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/˜rek/AEPStart.html;baserv.uci.kun.nl/˜jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/˜martin/abs/index.html; antibody.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/˜honegger/AHOseminar/Slide01.html;www.cryst.bbk.ac.uk/˜ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/˜fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html and in Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Dept.Health (1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR (framework) residues can be selected and combined from theconsensus and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. Humanization orengineering of antibodies of the present invention can be performedusing any known method, such as but not limited to those described in,Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323,5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323,5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101,5,585,089, 5,225,539; 4,816,567, PCT/: US98/16280, US96/18978,US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

The anti-alpha-V subunit antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-alpha-V subunit antibody can be isolated fromsuch animals and immortalized using suitable methods, such as themethods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol. 6(4) 579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90 (8) 3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

To generate fully human monoclonal antibodies to Alpha V, HuMAb mice canbe immunized with a purified or enriched preparation of Alpha V antigenand/or cells expressing Alpha V, as described by Lonberg, N. et al.(1994) Nature 3 68 (6474) 856.859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851 and WO 98/24884. Preferably, the mice will be6-16 weeks of age upon the first infusion. For example, a purified orenriched preparation (5-20 pg) of Alpha V antigen (e.g., purified fromAlpha V-expressing LNCaP cells) can be used to immunize the HuMAb miceintraperitoneally. In the event that immunizations using a purified orenriched preparation of Alpha V antigen do not result in antibodies,mice can also be immunized 1 5 with cells expressing Alpha V, e.g., atumor cell line, to promote immune responses. Cumulative experience withvarious antigens has shown that the HuMAb transgenic mice typicallyrespond best when initially immunized intraperitoneally (IP) withantigen in complete Freund's adjuvant, followed by every other week i.p.immunizations (up to a total of 6) with antigen in incomplete Freund'sadjuvant, followed by every other week IP/SC immunizations (up to atotal of 10) with antigen in incomplete Freund's adjuvant. The immuneresponse can be monitored over the course of the immunization protocolwith plasma samples being obtained by retroorbital bleeds. The plasmacan be screened by ELISA (as described below), and mice with sufficienttiters of anti-Alpha V human immunoglobulin can be used for fusions.Mice can be boosted intravenously with antigen 3 days before sacrificeand removal of the spleen. It is expected that 2-3 fusions for eachantigen may need to be performed. Several mice will be immunized foreach antigen.

To generate hybridomas producing human monoclonal antibodies to Alpha V,splenocytes and lymph node cells from immunized mice can be isolated andfused to an appropriate immortalized cell line, such as a mouse myelomacell line. The resulting hybridomas can be screened for the productionof antigen-specific antibodies.

Human antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification, site directed mutagenesis) and can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the VH segmentis operatively linked to the CH segment(s) within the vector and the VI,segment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino tem-finusof the antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (I 985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfrCHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:42164220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (I 982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for usewith NSO myeloma cells, another preferred expression system is the GSgene expression system disclosed in WO 87/04462, WO 89/01036 and EP338,841. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Screening antibodies for specific binding to similar proteins orfragments can also be conveniently achieved using peptide displaylibraries. This method involves the screening of large collections ofpeptides for individual members having the desired function orstructure. antibody screening of peptide display libraries is well knownin the art. The displayed peptide sequences can be from 3 to 5000 ormore amino acids in length, frequently from 5-100 amino acids long, andoften from about 8 to 25 amino acids long. In addition to directchemical synthetic methods for generating peptide libraries, severalrecombinant DNA methods have been described. One type involves thedisplay of a peptide sequence on the surface of a bacteriophage or cell.Each bacteriophage or cell contains the nucleotide sequence encoding theparticular displayed peptide sequence. Such methods are described in PCTPatent Publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278.Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Pat.Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, andscreening kits are commercially available from such suppliers asInvitrogen (Carlsbad, Calif.), and Cambridge antibody Technologies(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666,4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730,5,763,733, 5,767,260, 5,856,456, assigned to Enzon; 5,223,409,5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717,assigned to Affymax; 5,885,793, assigned to Cambridge antibodyTechnologies; 5,750,373, assigned to Genentech, 5,618,920, 5,595,898,5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan,supra; Ausubel, supra; or Sambrook, supra, each of the above patents andpublications entirely incorporated herein by reference.

Antibodies of the present invention can also be prepared using at leastone anti-alpha-V subunit antibody encoding nucleic acid to providetransgenic animals or mammals, such as goats, cows, horses, sheep, andthe like, that produce such antibodies in their milk. Such animals canbe provided using known methods. See, e.g., but not limited to, U.S.Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616;5,565,362; 5,304,489, and the like, each of which is entirelyincorporated herein by reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-alpha-V subunit antibody encoding nucleic acid toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco, maize, and duckweed) that produce such antibodies,specified portions or variants in the plant parts or in cells culturedtherefrom. As a non-limiting example, transgenic tobacco leavesexpressing recombinant proteins have been successfully used to providelarge amounts of recombinant proteins, e.g., using an induciblepromoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol.240:95-118 (1999) and references cited therein. Also, transgenic maizehave been used to express mammalian proteins at commercial productionlevels, with biological activities equivalent to those produced in otherrecombinant systems or purified from natural sources. See, e.g., Hood etal., Adv. Exp. Med. Biol. 464:127-147 (1999) and references citedtherein. antibodies have also been produced in large amounts fromtransgenic plant seeds including antibody fragments, such as singlechain antibodies (scFv's), including tobacco seeds and potato tubers.See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) andreference cited therein. Thus, antibodies of the present invention canalso be produced using transgenic plants, according to know methods. Seealso, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108(October, 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma etal., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc.Trans. 22:940-944 (1994); and references cited therein. See, alsogenerally for plant expression of antibodies, but not limited to, Eachof the above references is entirely incorporated herein by reference.

2. Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, specified fragments, variants orconsensus sequences thereof, or a deposited vector comprising at leastone of these sequences, a nucleic acid molecule of the present inventionencoding at least one anti-alpha-V subunit antibody can be obtainedusing methods described herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOS: 1-3) or light chain (e.g.,SEQ ID NOS: 4-6); nucleic acid molecules comprising the coding sequencefor an anti-alpha-V subunit antibody or variable region (e.g., SEQ IDNOS: 7, 8) including but not limited to SEQ ID Nos; 18 and 19; andnucleic acid molecules which comprise a nucleotide sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode at least one anti-alpha-Vsubunit antibody as described herein and/or as known in the art. Ofcourse, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate nucleicacid variants that code for specific anti-alpha-V subunit antibodies ofthe present invention. See, e.g., Ausubel, et al., supra, and suchnucleic acid variants are included in the present invention.Non-limiting examples of isolated nucleic acid molecules of the presentinvention include SEQ ID NOS: 10, 11, 12, 13, 14, 15, 18, and 19corresponding to non-limiting examples of a nucleic acid encoding,respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HCvariable region and LC variable region.

In another aspect, the invention provides isolated nucleic acidmolecules encoding a(n) anti-alpha-V subunit antibody having an aminoacid sequence as encoded by the nucleic acid contained in the plasmiddesignated clone C371A.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-alpha-V subunit antibodycan include, but are not limited to, those encoding the amino acidsequence of an antibody fragment, by itself; the coding sequence for theentire antibody or a portion thereof; the coding sequence for anantibody, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example—ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding an antibody can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedantibody comprising an antibody fragment or portion.

3. Polynucleotides which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

4. Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

5. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

6. Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 toInnis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 toGyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, etal; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al, with the tradename NASBA), the entire contents of whichreferences are incorporated herein by reference. (See, e.g., Ausubel,supra; or Sambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

7. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

8. Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (I 990). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withI 0 methotrexate selection/amplification) and the neo gene (for G418selection).

9. Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-alpha-V subunit antibody by recombinant techniques,as is well known in the art. See, e.g., Sambrook, et al., supra;Ausubel, et al., supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, PerC.6 cells, hep G2 cells,P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the like, which arereadily available from, for example, American Type Culture Collection,Manassas, Va. (www.atcc.org). Preferred host cells include cells oflymphoid origin such as myeloma and lymphoma cells.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenylation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenylationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art. Also, to avoid high surface expression of heavy chainmolecules, it may be necessary to use an expression vector thateliminates transmembrane domain variant splices.

10. Purification of an Antibody

An anti-alpha-V subunit antibody can be recovered and purified fromrecombinant cell cultures by well-known methods including, but notlimited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

11. Anti-Alpha-V Subunit Antibodies of the Invention

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention prepared as set forth above preferablycomprise the heavy and light chain CDR3s of CNTO 95. The antibodiesfurther can comprise the CDR2s of CNTO 95. The antibodies further cancomprise the CDR1s of CNTO 95. Accordingly, the invention furtherprovides anti-alpha V antibodies comprising: (1) human heavy chainframework regions, a human heavy chain CDR1 region, a human heavy chainCDR2 region, and a human heavy chain CDR3 region, wherein the humanheavy chain CDR3 region is selected from the CDR3s of CNTO 95 as shownin SEQ ID NO: 3, and (2) human light chain framework regions, a humanlight chain CDR1 region, a human light chain CDR2 region, and a humanlight chain CDR3 region, wherein the human light chain CDR3 region isselected from the CDR3s of CNTO 95 as shown in SEQ ID NO: 6, wherein theantibody binds Alpha V integrin. The antibody may further comprise theheavy chain CDR2 and/or the light chain CDR2 of CNTO 95. The antibodymay further comprise the heavy chain CDR1 and/or the light chain CDR1 ofCNTO 95.

As a non-limiting example, the antibody or antigen-binding portion orvariant can comprise at least one of the heavy chain CDR3 having theamino acid sequence of SEQ ID NO:3, and/or a light chain CDR3 having theamino acid sequence of SEQ ID NO:6. In a particular embodiment, theantibody or antigen-binding fragment can have an antigen-binding regionthat comprises at least a portion of at least one heavy chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 1, 2, and/or 3). Inanother particular embodiment, the antibody or antigen-binding portionor variant can have an antigen-binding region that comprises at least aportion of at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3)having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3(e.g., SEQ ID NOS: 4, 5, and/or 6). In a preferred embodiment the threeheavy chain CDRs and the three light chain CDRs of the antibody orantigen-binding fragment have the amino acid sequence of thecorresponding CDR of at least one of mAb CNTO 95, GenO101, CNTO 95,C372A, as described herein. Such antibodies can be prepared bychemically joining together the various portions (e.g., CDRs, framework)of the antibody using conventional techniques, by preparing andexpressing a (i.e., one or more) nucleic acid molecule that encodes theantibody using conventional techniques of recombinant DNA technology orby using any other suitable method.

Preferably, the CDR1, 2, and/or 3 of the engineered antibodies describedabove comprise the exact amino acid sequence(s) as those of CNTO 95disclosed herein. However, the ordinarily skilled artisan willappreciate that some deviation from the exact CDR sequences of CNTO 95may be possible while still retaining the ability of the antibody tobind Alpha V effectively (e.g., conservative substitutions).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of CNTO 95. In addition to simplybinding Alpha V, engineered antibodies such as those described above maybe selected for their retention of other functional properties ofantibodies of the invention, such as:

1). binding to live cells expressing human Alpha V; 2) binding to humanAlpha V with a K_(D) of 10⁻⁸ M or less (e.g., 10⁻⁹ M or 10^(−1O) M orless); 3) binding to a unique epitope on Alpha V (to eliminate thepossibility that monoclonal antibodies with complimentary activitieswhen used in combination would compete for binding to the same epitope);4) inhibition of angiogenesis resulting in growth inhibition of tumorcells in vivo.

Human monoclonal antibodies of the invention can be tested for bindingto Alpha V by, for example, standard ELISA.

To determine if the selected human anti-alpha V monoclonal antibodiesbind to unique epitopes, each antibody can be biotinylated usingcommercially available reagents (Pierce, Rockford, Ill.). Competitionstudies using unlabeled monoclonal antibodies and biotinylatedmonoclonal antibodies can be performed using alpha V coated-ELISAplates. Biotinylated mAb binding can be detected with astrep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed. In order to demonstrate binding of monoclonal antibodies tolive cells expressing the alpha V, flow cytometry can be used.Anti-alpha V human IgGs can be further tested for reactivity with alphaV antigen by Western blotting.

In another aspect of the invention, the structural features of an humananti-alpha V antibodies of the invention, CNTO 95, are used to createstructurally related human anti-Alpha V antibodies that retain at leastone functional property of the antibodies of the invention, such asbinding to Alpha V. More specifically, one or more CDR regions of CNTO95 can be combined recombinantly with known human framework regions andCDRs to create additional, recombinantly-engineered, human anti-Alpha Vantibodies of the invention.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-Alpha V antibody comprising: preparing an antibodycomprising (1) human heavy chain framework regions and human heavy chainCDRs, wherein at least one of the human heavy chain CDRs comprises anamino acid sequence selected from the amino acid sequences of CDRs shownin SEQ ID NOs: 1-3; and (2) human light chain framework regions andhuman light chain CDRs, wherein at least one of the human heavy chainCDRs comprises an amino acid-sequence selected from the amino acidsequences of CDRs shown in SEQ ID NOs: 4-6; wherein the antibody retainsthe ability to bind to Alpha V. The ability of the antibody to bindAlpha V can be determined using standard binding assays, such as thoseset forth in the Examples (e.g., an ELISA).

The antibodies of the invention can bind human alpha-V subunit with awide range of affinities (K_(D)). In a preferred embodiment, at leastone human mAb of the present invention can optionally bind human alpha-Vsubunit with high affinity. For example, a human mAb can bind humanalpha-V subunit with a K_(D) equal to or less than about 10⁻⁷ M, such asbut not limited to, 0.1-9.9 (or any range or value therein) X 10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M or any range or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W.H. Freeman and Company: New York, N.Y. (1992); and methodsdescribed herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Preferably, the human antibody or antigen-binding fragment of theinvention binds human alpha-V subunit and, thereby partially orsubstantially neutralizes at least one biological activity of theprotein. An antibody, or specified portion or variant thereof, thatpartially or preferably substantially neutralizes at least onebiological activity of at least one alpha-V subunit protein or fragmentcan bind the protein or fragment and thereby inhibit activities mediatedthrough the binding of alpha-V subunit to its ligand or through otheralpha-V subunit-dependent or mediated mechanisms. As used herein, theterm “neutralizing antibody” refers to an antibody that can inhibit analpha-V subunit-dependent activity by about 20-120%, preferably by atleast about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay. Thecapacity of an anti-alpha-V subunit antibody to inhibit an alpha-Vsubunit-dependent activity is preferably assessed by at least onesuitable alpha-V subunit protein or receptor assay, as described hereinand/or as known in the art. A human antibody of the invention can be ofany class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise akappa or lambda light chain. In one embodiment, the human antibodycomprises an IgG heavy chain or defined fragment, for example, at leastone of isotypes, IgG1, IgG2, IgG3 or IgG4. Antibodies of this type canbe prepared by employing a transgenic mouse or other transgenicnon-human mammal comprising at least one human light chain (e.g., IgG,IgA and IgM (e.g., γ1, γ2, γ3, γ4) transgenes as described herein and/oras known in the art. In another embodiment, the anti-human alpha-Vsubunit human antibody comprises an IgG1 heavy chain and a IgG1 lightchain.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one alpha-V subunit protein, subunit,fragment, portion or any combination thereof. The at least one epitopecan comprise at least one antibody binding region that comprises atleast one portion of said protein, which epitope is preferably comprisedof at least one extracellular, soluble, hydrophillic, external orcytoplasmic portion of said protein. The at least one specified epitopecan comprise any combination of at least one amino acid sequence of atleast 1-3 amino acids to the entire specified portion of contiguousamino acids of the SEQ ID NOS:9, 16 or 17.

As previously stated, the invention also relates to antibodies,antigen-binding fragments, immunoglobulin chains and CDRs comprisingamino acids in a sequence that is substantially the same as an aminoacid sequence described herein. Preferably, such antibodies orantigen-binding fragments and antibodies comprising such chains or CDRscan bind human alpha-V subunit with high affinity (e.g., K_(D) less thanor equal to about 10⁻⁹ M). Amino acid sequences that are substantiallythe same as the sequences described herein include sequences comprisingconservative amino acid substitutions, as well as amino acid deletionsand/or insertions. A conservative amino acid substitution refers to thereplacement of a first amino acid by a second amino acid that haschemical and/or physical properties (e.g, charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

An anti-alpha-V subunit antibody of the present invention can includeone or more amino acid substitutions, deletions or additions, eitherfrom natural mutations or human manipulation, as specified herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-alpha-V subunit antibody will not bemore than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-alpha-V subunit antibody of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one alpha-V subunit neutralizing activity. Sitesthat are critical for antibody binding can also be identified bystructural analysis such as crystallization, nuclear magnetic resonanceor photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904(1992) and de Vos, et al., Science 255:306-312 (1992)).

Anti-alpha-V subunit antibodies of the present invention can include,but are not limited to, at least one portion, sequence or combinationselected from 5 to all of the contiguous amino acids of at least one ofSEQ ID NOS:1, 2, 3, 4, 5, 6.

A(n) anti-alpha-V subunit antibody can further optionally comprise apolypeptide of at least one of 70-100% of the contiguous amino acids ofat least one of SEQ ID NOS: 7, 8.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS:7, 8. For example, theamino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO:8, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO:7. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 7, 8. The antibodies of the present invention,or specified variants thereof, can comprise any number of contiguousamino acid residues from an antibody of the present invention, whereinthat number is selected from the group of integers consisting of from10-100% of the number of contiguous residues in an anti-alpha-V subunitantibody. Optionally, this subsequence of contiguous amino acids is atleast about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more aminoacids in length, or any range or value therein. Further, the number ofsuch subsequences can be any integer selected from the group consistingof from 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-100% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5, 8, 11, 14-eicosatetraenoate (C₂₀,arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioicacid, docosanedioic acid, and the like. Suitable fatty acid estersinclude mono-esters of dicarboxylic acids that comprise a linear orbranched lower alkyl group. The lower alkyl group can comprise from oneto about twelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acryloyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphoramide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

12. Anti-Idiotype Antibodies to Anti-Alpha-V Subunit AntibodyCompositions

In addition to monoclonal or chimeric anti-alpha-V subunit antibodies,the present invention is also directed to an anti-idiotypic (anti-Id)antibody specific for such antibodies of the invention. An anti-Idantibody is an antibody which recognizes unique determinants generallyassociated with the antigen-binding region of another antibody. Theanti-Id can be prepared by immunizing an animal of the same species andgenetic type (e.g. mouse strain) as the source of the Id antibody withthe antibody or a CDR containing region thereof. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an anti-Id antibody. The anti-Idantibody may also be used as an “immunogen” to induce an immune responsein yet another animal, producing a so-called anti-anti-Id antibody.

13. Anti-Alpha-V Subunit Antibody Compositions

The present invention also provides at least one anti-alpha-V subunitantibody composition comprising at least one, at least two, at leastthree, at least four, at least five, at least six or more anti-alpha-Vsubunit antibodies thereof, as described herein and/or as known in theart that are provided in a non-naturally occurring composition, mixtureor form. Such compositions comprise non-naturally occurring compositionscomprising at least one or two full length, C- and/or N-terminallydeleted variants, domains, fragments, or specified variants, of theanti-alpha-V subunit antibody amino acid sequence selected from thegroup consisting of 70-100% of the contiguous amino acids of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, or specified fragments, domains or variantsthereof. Preferred anti-alpha-V subunit antibody compositions include atleast one or two full length, fragments, domains or variants as at leastone CDR or LBR containing portions of the anti-alpha-V subunit antibodysequence of 70-100% of SEQ ID NOS:1, 2, 3, 4, 5, 6, or specifiedfragments, domains or variants thereof. Further preferred compositionscomprise 40-99% of at least one of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5,6, or specified fragments, domains or variants thereof. Such compositionpercentages are by weight, volume, concentration, molarity, or molalityas liquid or dry solutions, mixtures, suspension, emulsions or colloids,as known in the art or as described herein.

Anti-alpha-V subunit antibody compositions of the present invention canfurther comprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-alpha-V subunit antibody to a cell, tissue, organ, animal orpatient in need of such modulation, treatment or therapy, optionallyfurther comprising at least one selected from at least one TNFantagonist (e.g., but not limited to a TNF antibody or fragment, asoluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a fluoroquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteroid, (dexamethasone), an anabolic steroid (testosterone), adiabetes related agent, a mineral, a nutritional, a thyroid agent, avitamin, a calcium related hormone, an antidiarrheal, an antitussive, anantiemetic, an antiulcer, a laxative, an anticoagulant, anerythropoietin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF,Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, animmunoglobulin (rituximab), an immunosuppressive (e.g., basiliximab,cyclosporine, daclizumab), a growth hormone, a hormone antagonist, areproductive hormone antagonist (flutamide, nilutamide), a hormonerelease modulator (leuprolide, goserelin), a hormone replacement drug,an estrogen receptor modulator (tamoxifen), a retinoid (tretinoin), atopoisomerase inhibitor (etoposide, irinotecan), a cytoxin (doxorubicin,dacarbazine), a mydriatic, a cycloplegic, an alkylating agent(carboplatin), a nitrogen mustard (melphalen, chlorabucil), anitrosourea (carmustine, estramustine) an antimetabolite (methotrexate,cytarabine, fluorouracil), a mitotic inhibitor (vincristine, taxol), aradiopharmaceutical (Iodine131-tositumomab), a radiosensitizer(misonidazole, tirapazamine) an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine(interferon alpha-2, IL2) or a cytokine antagonist (infliximab).Non-limiting examples of such cytokines include, but are not limited to,any of IL-1 to IL-23, IL-6, anti-tumor antibodies, chemotherapeuticagents or radiation therapies. Suitable dosages are well known in theart. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd)Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia,Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing,Loma Linda, Calif. (2000), each of which references are entirelyincorporated herein by reference.

Such anti-cancer can also include toxin molecules that are associated,bound, co-formulated or co-administered with at least one antibody ofthe present invention. The toxin can optionally act to selectively killthe pathologic cell or tissue. The pathologic cell can be a cancer orother cell. Such toxins can be, but are not limited to, purified orrecombinant toxin or toxin fragment comprising at least one functionalcytotoxic domain of toxin, e.g., selected from at least one of ricin,diphtheria toxin, a venom toxin, or a bacterial toxin. The term toxinalso includes both endotoxins and exotoxins produced by any naturallyoccurring, mutant or recombinant bacteria or viruses which may cause anypathological condition in humans and other mammals, including toxinshock, which can result in death. Such toxins may include, but are notlimited to, enterotoxigenic E. coli heat-labile enterotoxin (LT),heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonasenterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcalenterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins andthe like. Such bacteria include, but are not limited to, strains of aspecies of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli(e.g., strains of serotype 0157:H7), Staphylococcus species (e.g.,Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g.,Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigellasonnei), Salmonella species (e.g., Salmonella typhi, Salmonellacholera-suis, Salmonella enteritidis), Clostridium species (e.g.,Clostridium perfringens, Clostridium dificile, Clostridium botulinum),Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus),Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species(e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae),Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g.,Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species,Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed.,INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston,(1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiologyand Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York(1991); Mandell et al, Principles and Practice of Infectious Diseases,3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., TheMerck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood etal, FEMS Microbiology Immunology, 76:121-134 (1991); Marrack et al,Science, 248:705-711 (1990), the contents of which references areincorporated entirely herein by reference.

Anti-alpha-V subunit antibody compounds, compositions or combinations ofthe present invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-alpha-V subunit antibody, fragment orvariant composition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-alpha-V subunit antibody compositions can also include a buffer ora pH adjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-alpha-V subunit antibody compositions of theinvention can include polymeric excipients/additives such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-cyclodextrin), polyethyleneglycols, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids),steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-alpha-V subunit antibody, portion orvariant compositions according to the invention are known in the art,e.g., as listed in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), and in the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), thedisclosures of which are entirely incorporated herein by reference.Preferred carrier or excipient materials are carbohydrates (e.g.,saccharides and alditols) and buffers (e.g., citrate) or polymericagents.

14. Formulations

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one anti-alpha-V subunit antibody ina pharmaceutically acceptable formulation. Preserved formulationscontain at least one known preservative or optionally selected from thegroup consisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal(e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5,0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001,0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2,0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-alpha-V subunit antibody with theprescribed buffers and/or preservatives, optionally in an aqueousdiluent, wherein said packaging material comprises a label thatindicates that such solution can be held over a period of 1, 2, 3, 4, 5,6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.The invention further comprises an article of manufacture, comprisingpackaging material, a first vial comprising lyophilized at least oneanti-alpha-V subunit antibody, and a second vial comprising an aqueousdiluent of prescribed buffer or preservative, wherein said packagingmaterial comprises a label that instructs a patient to reconstitute theat least one anti-alpha-V subunit antibody in the aqueous diluent toform a solution that can be held over a period of twenty-four hours orgreater.

The at least one anti-alpha-V subunitantibody used in accordance withthe present invention can be produced by recombinant means, includingfrom mammalian cell or transgenic preparations, or can be purified fromother biological sources, as described herein or as known in the art.

The range of at least one anti-alpha-V subunit antibody in the productof the present invention includes amounts yielding upon reconstitution,if in a wet/dry system, concentrations from about 1.0 μg/ml to about1000 mg/ml, although lower and higher concentrations are operable andare dependent on the intended delivery vehicle, e.g., solutionformulations will differ from transdermal patch, pulmonary,transmucosal, or osmotic or micro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-alpha-V subunit antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-alpha-V subunit antibodyand preservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-alpha-V subunitantibody in buffered solution is combined with the desired preservativein a buffered solution in quantities sufficient to provide the proteinand preservative at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least oneanti-alpha-V subunit antibody that is reconstituted with a second vialcontaining water, a preservative and/or excipients, preferably aphosphate buffer and/or saline and a chosen salt, in an aqueous diluent.Either a single solution vial or dual vial requiring reconstitution canbe reused multiple times and can suffice for a single or multiple cyclesof patient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

The solutions of at least one anti-alpha-V subunit antibody in theinvention can be prepared by a process that comprises mixing at leastone antibody in an aqueous diluent. Mixing is carried out usingconventional dissolution and mixing procedures. To prepare a suitablediluent, for example, a measured amount of at least one antibody inwater or buffer is combined in quantities sufficient to provide theprotein and optionally a preservative or buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that can be optimizedfor the concentration and means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-alpha-Vsubunit antibody that is reconstituted with a second vial containing theaqueous diluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-alpha-V subunit antibody that is reconstituted with a second vialcontaining the aqueous diluent. The clear solution in this case can beup to one liter or even larger in size, providing a large reservoir fromwhich smaller portions of the at least one antibody solution can beretrieved one or multiple times for transfer into smaller vials andprovided by the pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,GenotropinPen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®,Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®,e.g., as made or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com). Recognized devices comprising a dual vial systeminclude those pen-injector systems for reconstituting a lyophilized drugin a cartridge for delivery of the reconstituted solution such as theHumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one anti-alpha-V subunitantibody in the aqueous diluent to form a solution and to use thesolution over a period of 2-24 hours or greater for the two vial,wet/dry, product. For the single vial, solution product, the labelindicates that such solution can be used over a period of 2-24 hours orgreater. The presently claimed products are useful for humanpharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-alpha-V subunit antibody and aselected buffer, preferably a phosphate buffer containing saline or achosen salt. Mixing the at least one antibody and buffer in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-alpha-V subunit antibody that is reconstituted with asecond vial containing a preservative or buffer and excipients in anaqueous diluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

At least one anti-alpha-V subunit antibody in either the stable orpreserved formulations or solutions described herein, can beadministered to a patient in accordance with the present invention via avariety of delivery methods including SC or IM injection; transdermal,pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump,or other means appreciated by the skilled artisan, as well-known in theart.

15. Therapeutic Applications

The anti-alpha-V subunit antibodies of the present invention orspecified variants thereof can be used to measure or effect in an cell,tissue, organ or animal (including mammals and humans), to diagnose,monitor, modulate, treat, alleviate, help prevent the incidence of, orreduce the symptoms of, at least one condition mediated, affected ormodulated by alpha V integrins. Such conditions are selected from, butnot limited to, diseases or conditions mediated by cell adhesion and/orangiogenesis. Such diseases or conditions include an immune disorder ordisease, a cardiovascular disorder or disease, an infectious, malignant,and/or neurologic disorder or disease, or other known or specifiedalpha-V integrin subunit related conditions. In particular, theantibodies are useful for the treatment of diseases that involveangiogenesis such as disease of the eye and neoplastic disease, tissueremodeling such as restenosis, and proliferation of certain cells typesparticularly epithelial and squamous cell carcinomas. Particularindications include use in the treatment of atherosclerosis, restenosis,cancer metastasis, rheumatoid arthritis, diabetic retinopathy andmacular degeneration. The neutralizing antibodies of the invention arealso useful to prevent or treat unwanted bone resorption or degradation,for example as found in osteoporosis or resulting from PTHrPoverexpression by some tumors. The antibodies may also be useful in thetreatment of various fibrotic diseases such as idiopathic pulmonaryfibrosis, diabetic nephropathy, hepatitis, and cirrhosis.

Thus, the present invention provides a method for modulating or treatingat least one alpha-V subunit related disease, in a cell, tissue, organ,animal, or patient, as known in the art or as described herein, using atleast one alpha-V subunit antibody of the present invention. Particularindications are discussed below:

Malignant Disease

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL,acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodysplasticsyndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, renal cellcarcinoma, breast cancer, nasopharyngeal carcinoma, malignanthistiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,solid tumors, adenocarcinomas, squamous cell carcinomas, sarcomas,malignant melanoma, particularly metastatic melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like.

Immune Related Disease

The present invention also provides a method for modulating or treatingat least one immune related disease, in a cell, tissue, organ, animal,or patient including, but not limited to, at least one of rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatorybowel disease, ulcerative colitis, systemic lupus erythematosis,antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis,idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitivity reactions, allergic rhinitis, hayfever, perennial rhinitis, conjunctivitis, endometriosis, asthma,urticaria, systemic anaphylaxis, dermatitis, pernicious anemia,hemolytic disease, thrombocytopenia, graft rejection of any organ ortissue, kidney transplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary biliary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemochromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, OKT3 therapy,anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g., theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al.,eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference.

Cardiovascular Disease

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic arteriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aortic andperipheral aneurysms, aortic dissection, inflammation of the aorta,occlusion of the abdominal aorta and its branches, peripheral vasculardisorders, occlusive arterial disorders, peripheral atheroscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-alpha-V subunit antibody to a cell, tissue,organ, animal or patient in need of such modulation, treatment ortherapy.

Neurologic Disease

The present invention also provides a method for modulating or treatingat least one neurologic disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit’ such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16^(th) Edition,Merck & Company, Rahway, N.J. (1992).

The present invention also provides a method for modulating or treatingat least one infectious disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: acute orchronic bacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-alpha-V subunit antibody to a cell, tissue,organ, animal or patient in need of such modulation, treatment ortherapy. Such a method can optionally further at least one selected fromat least one TNF antagonist (e.g., but not limited to a TNF antibody orfragment, a soluble TNF receptor or fragment, fusion proteins thereof,or a small molecule TNF antagonist), an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalazine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluoroquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod (dexamethasone), an anabolic steroid (testosterone), adiabetes related agent, a mineral, a nutritional, a thyroid agent, avitamin, a calcium related hormone, an antidiarrheal, an antitussive, anantiemetic, an antiulcer, a laxative, an anticoagulant, anerythropoietin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF,Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, animmunoglobulin (rituximab), an immunosuppressive (e.g., basiliximab,cyclosporine, daclizumab), a growth hormone, a hormone antagonist, areproductive hormone antagonist (flutamide, nilutamide), a hormonerelease modulator (leuprolide, goserelin), a hormone replacement drug,an estrogen receptor modulator (tamoxifen), a retinoid (tretinoin), atopoisomerase inhibitor (etoposide, irinotecan), a cytoxin(doxorubicin), a mydriatic, a cycloplegic, an alkylating agent(carboplatin), a nitrogen mustard (melphalen, chlorabucil), anitrosourea (carmustine, estramustine) an antimetabolite (methotrexate,cytarabine, fluorouracil), a mitotic inhibitor (vincristine, taxol), aradiopharmaceutical (Iodine131-tositumomab), a radiosensitizer(misonidazole, tirapazamine) an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine(interferon alpha-2, IL2) or a cytokine antagonist (infliximab).Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Particular combinations for treatment of neoplastic diseases compriseco-administration or combination therapy by administering, beforeconcurrently, and/or after, an antineoplastic agent such as analkylating agent, a nitrogen mustard, a nitrosurea, an antibiotic, ananti-metabolite, a hormonal agonist or antagonist, an immunomodulator,and the like. For use in metastatic melanoma and other neoplasticdiseases, a preferred combination is to co-administer the antibody withdacarbazine, interferon alpha, interleukin-2, temozolomide, cisplatin,vinblastine, Imatinib Mesylate, carmustine, paclitaxel and the like. Formetastatic melanoma, dacarbazine is preferred.

Therapeutic Treatments

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-alpha-Vsubunit antibody composition that total, on average, a range from atleast about 0.01 to 500 milligrams of at least one anti-alpha-Vsubunitantibody per kilogram of patient per dose, and preferably from atleast about 0.1 to 100 milligrams antibody kilogram of patient persingle or multiple administration, depending upon the specific activityof contained in the composition. Alternatively, the effective serumconcentration can comprise 0.1-5000 μg/ml serum concentration per singleor multiple administration. Suitable dosages are known to medicalpractitioners and will, of course, depend upon the particular diseasestate, specific activity of the composition being administered, and theparticular patient undergoing treatment. In some instances, to achievethe desired therapeutic amount, it can be necessary to provide forrepeated administration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes of can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one anti-alpha-V subunit antibody according to the presentinvention. While pulmonary administration is used in the followingdescription, other modes of administration can be used according to thepresent invention with suitable results.

Alpha-V subunit antibodies of the present invention can be delivered ina carrier, as a solution, emulsion, colloid, or suspension, or as a drypowder, using any of a variety of devices and methods suitable foradministration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aqueous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthetic mono- or di- or tri-glycerides. Parental administration isknown in the art and includes, but is not limited to, conventional meansof injections, a gas pressured needle-less injection device as describedin U.S. Pat. No. 5,851,198, and a laser perforator device as describedin U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.

Alternative Delivery

The invention further relates to the administration of at least oneanti-alpha-V subunit antibody by parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermalmeans. At least one anti-alpha-V subunit antibody composition can beprepared for use for parenteral (subcutaneous, intramuscular orintravenous) or any other administration particularly in the form ofliquid solutions or suspensions; for use in vaginal or rectaladministration particularly in semisolid forms such as, but not limitedto, creams and suppositories; for buccal, or sublingual administrationsuch as, but not limited to, in the form of tablets or capsules; orintranasally such as, but not limited to, the form of powders, nasaldrops or aerosols or certain agents; or transdermally such as notlimited to a gel, ointment, lotion, suspension or patch delivery systemwith chemical enhancers such as dimethyl sulfoxide to either modify theskin structure or to increase the drug concentration in the transdermalpatch (Junginger, et al. In “Drug Permeation Enhancement”; Hsieh, D. S.,Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirelyincorporated herein by reference), or with oxidizing agents that enablethe application of formulations containing proteins and peptides ontothe skin (WO 98/53847), or applications of electric fields to createtransient transport pathways such as electroporation, or to increase themobility of charged drugs through the skin such as iontophoresis, orapplication of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989and 4,767,402) (the above publications and patents being entirelyincorporated herein by reference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably at least one anti-alpha-Vsubunit antibody composition is delivered in a particle size effectivefor reaching the lower airways of the lung or sinuses. According to theinvention, at least one anti-alpha-V subunit antibody can be deliveredby any of a variety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of antibodies are alsoknown in the art. All such devices can use of formulations suitable forthe administration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles. Metered dose inhalers like the Ventolin®metered dose inhaler, typically use a propellent gas and requireactuation during inspiration (See, e.g., WO 94/16970, WO 98/35888). Drypowder inhalers like Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus®(Glaxo), Spiros™ inhaler (Dura), devices marketed by InhaleTherapeutics, and the Spinhaler® powder inhaler (Fisons), usebreath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra, EP237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein byreference). Nebulizers like AERx™ Aradigm, the Ultravent® nebulizer(Mallinckrodt), and the Acorn II® nebulizer (Marquest Medical Products)(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above referencesentirely incorporated herein by reference, produce aerosols fromsolutions, while metered dose inhalers, dry powder inhalers, etc.generate small particle aerosols. These specific examples ofcommercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of thisinvention, and are not intended as limiting the scope of the invention.Preferably, a composition comprising at least one anti-alpha-V subunitantibody is delivered by a dry powder inhaler or a sprayer. There are aseveral desirable features of an inhalation device for administering atleast one antibody of the present invention. For example, delivery bythe inhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g. less than about 10 m, preferably about 1-5 m, for goodrespirability.

Administration of Alpha-V Subunit Antibody Compositions as a Spray

A spray including alpha-V subunit antibody composition protein can beproduced by forcing a suspension or solution of at least oneanti-alpha-V subunit antibody through a nozzle under pressure. Thenozzle size and configuration, the applied pressure, and the liquid feedrate can be chosen to achieve the desired output and particle size. Anelectrospray can be produced, for example, by an electric field inconnection with a capillary or nozzle feed. Advantageously, particles ofat least one anti-alpha-V subunit antibody composition protein deliveredby a sprayer have a particle size less than about 10 m, preferably inthe range of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

Formulations of at least one anti-alpha-V subunit antibody compositionprotein suitable for use with a sprayer typically include antibodycomposition protein in an aqueous solution at a concentration of about0.1 mg to about 100 mg of at least one anti-alpha-V subunit antibodycomposition protein per ml of solution or mg/gm, or any range or valuetherein, e.g., but not limited to, 0.1, 0.2., 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80,90 or 100 mg/ml or mg/gm. The formulation can include agents such as anexcipient, a buffer, an isotonicity agent, a preservative, a surfactant,and, preferably, zinc. The formulation can also include an excipient oragent for stabilization of the antibody composition protein, such as abuffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating antibody composition proteins includealbumin, protamine, or the like. Typical carbohydrates useful informulating antibody composition proteins include sucrose, mannitol,lactose, trehalose, glucose, or the like. The antibody compositionprotein formulation can also include a surfactant, which can reduce orprevent surface-induced aggregation of the antibody composition proteincaused by atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbitol fatty acidesters. Amounts will generally range between 0.001 and 14% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan monooleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein such as alpha-V subunit antibodies, orspecified portions or variants, can also be included in the formulation.

Administration of Alpha-V Subunit Antibody Compositions by a Nebulizer

Antibody composition protein can be administered by a nebulizer, such asjet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer,a compressed air source is used to create a high-velocity air jetthrough an orifice. As the gas expands beyond the nozzle, a low-pressureregion is created, which draws a solution of antibody compositionprotein through a capillary tube connected to a liquid reservoir. Theliquid stream from the capillary tube is sheared into unstable filamentsand droplets as it exits the tube, creating the aerosol. A range ofconfigurations, flow rates, and baffle types can be employed to achievethe desired performance characteristics from a given jet nebulizer. Inan ultrasonic nebulizer, high-frequency electrical energy is used tocreate vibrational, mechanical energy, typically employing apiezoelectric transducer. This energy is transmitted to the formulationof antibody composition protein either directly or through a couplingfluid, creating an aerosol including the antibody composition protein.Advantageously, particles of antibody composition protein delivered by anebulizer have a particle size less than about 10 μm, preferably in therange of about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm.

Formulations of at least one anti-alpha-V subunit antibody suitable foruse with a nebulizer, either jet or ultrasonic, typically include aconcentration of about 0.1 mg to about 100 mg of at least oneanti-alpha-V subunit antibody protein per ml of solution. Theformulation can include agents such as an excipient, a buffer, anisotonicity agent, a preservative, a surfactant, and, preferably, zinc.The formulation can also include an excipient or agent for stabilizationof the at least one anti-alpha-V subunit antibody composition protein,such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.Bulk proteins useful in formulating at least one anti-alpha-V subunitantibody composition proteins include albumin, protamine, or the like.Typical carbohydrates useful in formulating at least one anti-alpha-Vsubunit antibody include sucrose, mannitol, lactose, trehalose, glucose,or the like. The at least one anti-alpha-V subunit antibody formulationcan also include a surfactant, which can reduce or preventsurface-induced aggregation of the at least one anti-alpha-V subunitantibody caused by atomization of the solution in forming an aerosol.Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbital fatty acid esters. Amounts will generally range between 0.001and 4% by weight of the formulation. Especially preferred surfactantsfor purposes of this invention are polyoxyethylene sorbitan mono-oleate,polysorbate 80, polysorbate 20, or the like. Additional agents known inthe art for formulation of a protein such as antibody protein can alsobe included in the formulation.

Administration of Alpha-V Subunit Antibody Compositions by a MeteredDose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one anti-alpha-Vsubunit antibody, and any excipients or other additives are contained ina canister as a mixture including a liquefied compressed gas. Actuationof the metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably about 1 μm to about 5 μm, and most preferably about 2 μm toabout 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition protein produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one anti-alpha-V subunit antibody for use witha metered-dose inhaler device will generally include a finely dividedpowder containing at least one anti-alpha-V subunit antibody as asuspension in a non-aqueous medium, for example, suspended in apropellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-alpha-V subunit antibody as asuspension in the propellant, to protect the active agent againstchemical degradation, and the like. Suitable surfactants includesorbitan trioleate, soya lecithin, oleic acid, or the like. In somecases solution aerosols are preferred using solvents such as ethanol.Additional agents known in the art for formulation of a protein such asprotein can also be included in the formulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one anti-alpha-V subunit antibody compositions via devices notdescribed herein.

Oral Formulations and Administration

Formulations for oral rely on the co-administration of adjuvants (e.g.,resorcinols and nonionic surfactants such as polyoxyethylene oleyl etherand n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 are used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods ofadministering at least one anti-alpha-V subunit antibody include anemulsion comprising a plurality of submicron particles, a mucoadhesivemacromolecule, a bioactive peptide, and an aqueous continuous phase,which promotes absorption through mucosal surfaces by achievingmucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucoussurfaces suitable for application of the emulsions of the presentinvention can include corneal, conjunctival, buccal, sublingual, nasal,vaginal, pulmonary, stomachic, intestinal, and rectal routes ofadministration. Formulations for vaginal or rectal administration, e.g.suppositories, can contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor intranasal administration can be solid and contain as excipients,for example, lactose or can be aqueous or oily solutions of nasal drops.For buccal administration excipients include sugars, calcium stearate,magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No.5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the at least one anti-alpha-V subunitantibody is encapsulated in a delivery device such as a liposome orpolymeric nanoparticles, microparticle, microcapsule, or microspheres(referred to collectively as microparticles unless otherwise stated). Anumber of suitable devices are known, including microparticles made ofsynthetic polymers such as polyhydroxy acids such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

16. Diagnostic and Research Applications.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁶S, or ¹²⁶I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g.,¹²⁵I, ³²P, ¹⁴C, technicium, or ³H, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, e at., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be alpha.v or an immunologically reactive portion thereof) tocompete with the test sample analyte (alpha.v) for binding with alimited amount of antibody. The amount of .alpha.v in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent or radioisotope is administered to, a host,preferably into the bloodstream, and the presence and location of thelabeled antibody in the host is assayed. This imaging technique isuseful in the staging and treatment of neoplasms or bone disorders. Theantibody may be labeled with any moiety that is detectable in a host,whether by nuclear magnetic resonance, radiology, or other detectionmeans known in the art.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1 Cloning and Expression of Alpha-V Subunit Antibody inMammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of alpha-V subunit antibody.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). The plasmid contains the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (e.g., alpha minusMEM, Life Technologies, Gaithersburg, Md.) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., F. W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L.Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M.J. Page and M. A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grownin increasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach can be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained that contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express thealpha-V subunit antibody in a regulated way in mammalian cells (M.Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).For the polyadenylation of the mRNA other signals, e.g., from the humangrowth hormone or globin genes can be used as well. Stable cell linescarrying a gene of interest integrated into the chromosomes can also beselected upon co-transfection with a selectable marker such as gpt, G418or hygromycin. It is advantageous to use more than one selectable markerin the beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete alpha-V subunit antibody is used,corresponding to HC and LC variable regions of a alpha-V subunitantibody of the present invention, according to known method steps.Isolated nucleic acid encoding a suitable human constant region (i.e.,HC and LC regions) is also used in this.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM.Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 2 Method of Making and Characterization of Non-Limiting Exampleof Fully Human Alpha-V Subunit Antibody

Summary. (CBA/J×C57/BL6/J) F₂ hybrid mice (Taylor et al., InternationalImmunology 6:579-591 (1993); Lonberg et al., Nature 368:856-859 (1994);Neuberger, Nature Biotechnology 14:826 (1996); Fishwild et al., NatureBiotechnology 14:845-851 (1996)) containing human variable and constantregion antibody transgenes for both heavy and light chains wereimmunized with human placental αVβ3. One fusion yielded 2 totally humanαVβ3 reactive IgG1κ monoclonal antibodies, named CNTO 95 and GenO.101.The totally human anti-αVβ3 antibodies were further characterized andboth were found to be reactive to the αVβ3 and αVβ5 subunits suggestingspecificity for the shared alpha chain of both molecules. One Mab, CNTO95, also known as CNTO 95, inhibits the binding of both αVβ3 and αVβ5 tovitronectin in cell based assays.

Abbreviations

-   -   BSA—bovine serum albumin    -   CO₂—carbon dioxide    -   DMSO—dimethyl sulfoxide    -   EIA—enzyme immunoassay    -   FBS—fetal bovine serum    -   H₂O₂—hydrogen peroxide    -   HC—heavy chain    -   HRP—horseradish peroxidase    -   Ig—immunoglobulin    -   IP—intraperitoneal    -   IV—intravenous    -   Mab—monoclonal antibody    -   OD—optical density    -   OPD—o-Phenylenediamine dihydrochloride    -   PEG—polyethylene glycol    -   PSA—penicillin, streptomycin, amphotericin    -   RT—room temperature    -   SQ—subcutaneous    -   TBS—Tris buffered saline    -   v/v—volume per volume    -   w/v—weight per volume

Introduction:

We have utilized transgenic mice that contain human heavy and lightchain immunoglobulin genes to generate totally human monoclonalantibodies that are specific to the αV integrins. These novel antibodiescan be used therapeutically to inhibit the angiogenic process byblocking the binding of αV integrins to their respective ECM ligands andprovide additional tools in the treatment of various cancers.

Materials and Methods

Animals

Transgenic mice have been developed by GenPharm International thatexpress human immunoglobulins but not mouse IgM or Igκ. These micecontain human sequence transgenes that undergo V(D)J joining,heavy-chain class switching and somatic mutation to generate arepertoire of human sequence immunoglobulins (Taylor et al.,International Immunology 6:579-591 (1993)). The light chain transgene isderived in part from a yeast artificial chromosome clone that includesnearly half of the germline human Vκ region. In addition to several VHgenes, the heavy-chain (HC) transgene encodes both human μ and human γ1(Lonberg et al., Nature 368:856-859 (1994)) and/or γ3 constant regions.A mouse derived from the HC012 genotypic lineage was used in theimmunization and fusion process to generate these monoclonal antibodies.

Purification of Human αVβ3

Human placenta (disrupted using a meat grinder) or M21 human melanomacells expressing the αVβ3 integrin were extracted with saline containing20 mM Tris pH 7.5, 1 mM CaCl₂, 1 mM MnCl₂, 100 mM Octylthioglucoside(OTG from Pierce), 0.05% sodium azide and 1 mM phenylmethylsulfonylfluoride (Sigma). The mixture was stirred for 1 hr at room temperatureand clarified by centrifugation at 10,000×g. The supernatant fromplacental extracts was applied to an affinity column consisting of Mab10E5 coupled to sepharose (Pharmacia) to remove GPIIb/IIIa and theflow-through fraction was applied to an affinity column consisting ofMab c7E3 Fab coupled to sepharose (Pharmacia) to bind αVβ3. The c7E3column was washed with PBS containing 1 mM CaCl₂, 1 mM MnCl₂, and 0.1%OTG followed by 0.1M sodium acetate pH 4.5, 1 mM CaCl₂, 1 mM MnCl₂, and0.1% OTG, pH 3.0. The column was eluted with 0.1M glycine, 2% aceticacid, 1 mM CaCl₂, 1 mM MnCl₂, and 0.1% OTG. The eluate containingpurified αVβ3 was neutralized using 2M Tris pH 8.5. Purity of thepreparations was characterized by SDS-PAGE analysis and ELISA to ruleout GPIIb/IIIa contamination (Wayner, et al., J. Cell Biol. 113: 919-929(1991)).

Immunizations

A fifteen to 17 week old surgically castrated male mouse obtained fromGenPharm was immunized IP (200 μL) and in 2 sites SQ (100 μL per site)with a total of 20 μg of placental αVβ3 (prep V fraction, JG21197)emulsified with an equal volume of complete Freund's adjuvant (day 0).The mouse was immunized two weeks later in the same manner with αVβ3emulsified with an equal volume of incomplete Freund's adjuvant. Threesubsequent 10 μg IP/10 μg SQ injections with incomplete Freund'sadjuvant were administered on days 28, 42, and 56. The mouse was thenbled on days 42 and 56 by retro-orbital puncture without anti-coagulant.The blood was allowed to clot at RT for one hour and the serum wascollected and titered using an αVβ3 solid phase EIA assay. The fusion,named GenO, was performed when repeated injections did not cause titersto increase. At that time, the mouse with a specific human IgG titer of1:1280 against αVβ3 was given a final IV booster injection of 10 μg αVβ3diluted in 100 μL physiological saline. Three days later, the mouse waseuthanized by cervical dislocation and the spleen was removedaseptically and immersed in 10 mL of cold phosphate buffered saline(PBS) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25μg/mL amphotericin B (PSA). The splenocytes were harvested by sterilelyperfusing the spleen with PSA-PBS. The cells were washed once in coldPSA-PBS, counted using Trypan blue dye exclusion and resuspended in RPMI1640 media containing 25 mM Hepes.

Cell Lines

The non-secreting mouse myeloma fusion partner, SP2/0 was employed. Thecell line was expanded in αMEM (modified) medium (JRH Biosciences)supplemented with 10% (v/v) FBS (Cell Culture Labs), 1 mM sodiumpyruvate, 0.1 mM NEAA, 2 mM L-glutamine (all from JRH Biosciences) andcryopreserved in 95% FBS and 5% DMSO (Sigma), then stored in a vaporphase liquid nitrogen freezer in CBS. The cell bank was sterile (QualityControl Centocor, Malvern) and free of mycoplasma (BioniqueLaboratories). Cells were maintained in log phase culture until fusion.They were washed in PBS, counted, and viability determined (>95%) viatrypan blue dye exclusion prior to fusion.

The M21 cell line, a human melanoma expressing the αVβ3 and αVβ5integrins, was expanded and cryopreserved. The 10-vial research cellbank was received into Cell Biology Services and stored in liquidnitrogen. The cell bank was sterile and free of mycoplasma (BioniqueLaboratories). The MDAMB435L2 cell line, a human breast carcinoma, was agift from Dr. Janet Price (MD Anderson, Houston Tex.) expresses theintegrin αVβ3. The cell line was cryopreserved in Cell Biology Services.The cell bank was sterile and free of mycoplasma (BioniqueLaboratories). M21 and MDAMB435L2 cells were thawed, propagated inappropriate media and maintained in log phase for several days prior touse in bioassays or allowed to reach confluency for use in thepurification of αVβ3 protein (M21 cells).

Cell Fusion

Fusion was carried out at a 1:1 ratio of murine myeloma cells (SP2/0) toviable spleen cells. Briefly, spleen cells and myeloma cells werepelleted together. The pellet was slowly resuspended, over 30 seconds,in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight 3,000,Sigma) at 37° C. The fusion was stopped by slowly adding 1 mL ofDulbecco's PBS (JRH) (37° C.) over 1 minute. An additional 19 mL of PBSwas added over the next 90 seconds. The fused cells were centrifuged for5 minutes at 750 rpm. The cells were then resuspended in HAT medium(αMEM medium containing 20% Fetal Bovine Serum (JRH), 1 mM sodiumpyruvate, 2 mM L-glutamine, 0.1 mM Non-essential amino acids, 10 μg/mLgentamicin, 2.5% Origen culturing supplement (Fisher), 50 μM2-mercaptoethanol, 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μMthymidine) and then plated at 200 μL/well in thirteen 96-well flatbottom tissue culture plates. The plates were then placed in ahumidified 37° C. incubator containing 5% CO₂ and 95% air for 7-10 days.

Detection of Human IgG Anti-αVβ3 Antibodies in Mouse Serum

Solid phase EIAs were used to screen mouse sera for human IgG antibodiesspecific for human αVβ3. Briefly, plates were coated with αVβ3 at 1μg/mL in PBS overnight. After washing in 0.15M saline containing 0.02%(v/v) Tween 20, the wells were blocked with 1% (w/v) BSA in HBSS withCa⁺⁺ and Mg⁺⁺, 200 μL/well for 1 hour at RT. Plates were usedimmediately or frozen at −20° C. for future use. Mouse sera wereincubated in doubling dilutions on the αVβ3 coated plates at 50 μL/wellat RT for 1 hour. The plates were washed and then probed with 50 μL/wellHRP-labeled goat anti-human IgG, Fc specific (Accurate) diluted 1:30,000in 1% BSA-PBS for 1 hour at RT. The plates were again washed and 100μL/well of the citrate-phosphate substrate solution (0.1M citric acidand 0.2M sodium phosphate, 0.01% H₂O₂ and 1 mg/mL OPD) was added for 15minutes at RT. Stop solution (4N sulfuric acid) was then added at 25μL/well and the OD's were read at 490 nm via an automated platespectrophotometer.

Detection of Totally Human Immunoglobulins in Hybridoma Supernatants

Because the GenPharm mouse is capable of generating both mouse and humanimmunoglobulin chains, growth positive hybridomas secreting fully humanimmunoglobulins were detected using two separate EIA systems. Plateswere coated as described above and undiluted hybridoma supernatants wereincubated on the plates for one hour at 37° C. The plates were washedand probed with either HRP labeled goat anti-human kappa (SouthernBiotech) antibody diluted 1:10,000 in 1% BSA-HBSS or HRP labeled goatanti-human IgG Fc specific antibody diluted to 1:30,000 in 1% BSA-HBSSfor one hour at 37° C. The plates were then incubated with substratesolution as described above.

Isotyping

Isotype determination of the antibodies was accomplished using an EIA ina format similar to that used to screen the mouse immune sera forspecific titers. αVβ3 was coated on 96-well plates as described aboveand purified antibody at 2 μg/mL was incubated on the plate for one hourat RT. The plate was washed and probed with HRP labeled goat anti-humanIgG₁ (Binding Site) or HRP labeled goat anti-human IgG₃ diluted at1:4000 (Zymed) in 1% BSA-HBSS for one hour at RT. The plate was againwashed and incubated with substrate solution as described above.

Preparation of Anti-Idiotype Antibodies to CNTO95

Seventeen monoclonal antibodies were made to the variable region of CNTO95. Six of them are non-blocking. The remaining eleven appear to blockthe active site of CNTO 95 and inhibit the binding of CNTO 95 to humanintegrin aVb3 and do not bind to CNTO 95 prebound to its receptor. Thenon-blocking Mab, CNTO 1073, can detect CNTO 95 pre-bound to aVb3.Pooled human serum does not interfere with the binding of 16 of 17 CNTO95 anti-variable region Mabs.

The anti-idiotype Mabs are useful in pharmacokinetic orimmunohistochemical detection of CNTO 95 in patient and animal tissue orsera samples as well as in epitope mapping efforts to define the bindingregions of CNTO 95 to its targe

Binding Characteristics of Human Monoclonal Antibodies to AlphaV by EIA

Binding characteristics for the antibodies were assessed using an αVβ3capture EIA. Linbro plates were coated with αVβ3 at 1 μg/mL in TBS with2 mM calcium overnight at 4° C. Plates were washed and blocked withTBS/1% BSA/calcium for at least one hour at room temperature. Purifiedantibodies were incubated in doubling dilutions from a startingconcentration of 2 μg/mL. Plates were washed and conjugated antibodies(HRP-labeled goat anti-human IgG Fc at 1:30,000) were added andincubated on plates for one hour at room temperature. Plates were washedOPD substrate was added to wells. Plates were read via an automatedplate spectrophotometer.

Competition of Binding of CNTO95 to M21 Cells by Various CommercialAnti-Integrin Mabs

M21 Cells were trypsinized from culture flasks, washed and resuspendedin HBSS/calcium to 2×10⁶ cell/mL. GenO95 was prelabeled with FITC-goatanti-human Fc (Jackson) for 30 minutes at RT. 10× concentrations ofGenO95 of 200 μg/mL or 20 μg/mL were incubated with FITC-goat anti-humanIgG at 250 μg/mL. Aliquots of 100 μL of M21 cells (2×10⁵ cells) wereincubated with 12 μL 10× GenO95 at high (20 μg/mL final) and low (2μg/mL final) concentrations±12 μL of the following murine antibodies:m7E3 IgG, anti-αVβ3 (clone LM609, Chemicon), anti-αVβ5 (clone P1F6,Gibco), anti-β3 (Chemicon, AMAC), or anti-αV (clone VNR139, Gibco)antibodies (at 20 μg/mL) for 45 minutes at 37° C. An aliquot was removedfrom each tube (for two-color analysis) and the remainder was fixed with1% paraformaldehyde and analyzed on a flow cytometer. For two-coloranalysis, an aliquot (50 μL) was incubated with PE-goat anti-mouse IgGfor 30 minutes at RT to label murine anti-αVβ3, anti-αVβ3, anti-β3, oranti-αV antibodies for two-color analysis. All tubes were fixed with 1%paraformaldehyde.

Inhibition of αVβ3 or αVβ5 Dependent M21 Cell or MDA MB435L2 CellAdhesion to Vitronectin Coated Plates by αVβ3/αVβ5 Specific Mabs

Linbro plates were coated for 1 hour at room temperature 50 μL/well ofvitronectin (Collaborative, Becton Dickinson) at 5 μg/mL in TBS with 2mM calcium. Plates were washed with HBSS/calcium and blocked with TBScontaining 2 mM calcium and 1% BSA for 30 minutes at RT. M21 cells weretrypsinized, washed once with media containing FCS and resuspended in 3mL HBSS without calcium. All washes were done with 10 minute spins at1000 rpm in the Sorvall tabletop centrifuge. To fluorescently label thecells, calcein (Molecular Probes) (5 mg/mL in DMSO) was added to thecells to a final concentration of 100 μg/mL in a 50 mL conical tube(wrapped in foil). Cells were incubated 10 to 15 minutes at 37° C.Calcein labeled cells were washed once with HBSS and resuspended in HBSSsupplemented with 0.1% BSA and 1 mM MgCl₂. Antibodies were titrated(14-fold dilution series) in HBSS/0.1% BSA/2 mM calcium at 10× finalconcentration. Cells (300 μL at 7.5×10⁶/mL) were preincubated withantibody titrations (37 μL of 10× solution)±anti-αVβ5 (P1F6) ascites(Chemicon) (37 μL of 1:600 (10×)) for 15 min at 37° C. The cell-antibodymixture was added to the vitronectin-coated plates at 100 μL/well intriplicate (approximately 6×10⁵ cells/well). Plates were incubated for45 minutes at 37° C. Unbound cells were removed by two washes withHBSS/calcium (150 μL/well). 100 μl HBSS/calcium was added to each welland the plate read on the Fluoroskan at 485-538 nm.

In a separate assay, MDA-MB435-L2 human breast carcinoma cells wereharvested with versene and suspended in serum free media at 500,000cells/mL and incubated with various concentrations of GenO95. After 10minutes of incubation tumor cell suspension (100 μL) was added tovitronectin (10 μg/mL) coated Linbro plates and incubated at 37° C.After 1 hour, wells were washed three times with serum free media (200μL/wash) and the MTT based Cell Titer AQ dye (Promega, Madison, Wis.)was added to each well. Extent of cell adhesion was determined in anELISA plate reader where OD490 nm is directly proportional to celladhesion. Cell adhesion to BSA coated wells served as negative control.

Determination of Ca⁺⁺ Dependence for Binding of Anti-HumanalphaVbeta3/alphaVbeta5 Mabs to their Ligands

To determine cation dependence in the binding of CNTO 95 and C372 toα_(v)β₃ or α_(v)β₅, a liquid phase EIA was utilized. EIA plates(Corning) were coated with CNTO 95, C372, c7E3 or LM609 IgG Mabs at 10:g/mL in carbonate coating buffer overnight at 4EC. Plates were blockedwith 1% BSA diluted in HBSS in the presence or absence of 2 mM Ca⁺⁺ forat least one hour at 37EC. Doubling dilutions of alphaVbeta3 (logJG52599) or alphaVbeta5 (Chemicon) starting at 10: g/mL werepreincubated with 50 mM EDTA (Sigma) in 1% BSA/HBSS without Ca⁺⁺ or with1% BSA/HBSS with Ca⁺⁺ for 30 minutes at 37° C. The mixtures were thenadded to the plates and incubated for 30 minutes at 37° C. The plateswere then washed and non-competing Mabs were added to the plates asfollows: to the CNTO 95, C372, c7E3 coated plates to detect alphaVbeta3binding, Mab LM609 was added at 20 microgm/mL in 1% BSA/HBSS Ca⁺⁺; tothe LM609 coated plate to detect alphaVbeta3 binding, Mab CNTO 95 wasadded at 20: g/mL in 1% BSA/HBSS w Ca⁺⁺ to the CNTO 95, C372, c7E3coated plates to detect alphaVbeta5 binding Mab VNR139 (Gibco) was addedat 10 microg/mL in 1% BSA/HBSS w Ca⁺⁺ and incubated for 30 minutes at37° C. The plates were again washed and probed with either HRP labeledgoat anti-mouse IgG Fc or HRP labeled goat anti-human IgG Fc inappropriate buffer and incubated for 30 minutes at 37° C. The plateswere washed, OPD substrate was added and the OD 490 measured aspreviously described.

Results and Discussion

Generation of Totally Human Anti-Human αVβ3 Integrin MonoclonalAntibodies

One fusion, named GenO, was performed from a GenPharm mouse immunizedwith alphaVbeta3 protein. From this fusion, 129 growth positive hybridswere screened. Two hybridoma cell lines were identified that secretedtotally human IgG antibodies reactive with human alphaVbeta3. These twocell lines, CNTO 95.9.12 and GenO.101.17.22, each secreteimmunoglobulins of the human IgG1κ isotype and both were subcloned twiceby limiting dilution to obtain stable cell lines (>90% homogeneous).CNTO 95.9.12 was assigned C-code #C371A and GenO.101.17.22 was assignedC-code #C372A. Each of the cell lines was frozen in 12-vial researchcell banks stored in LN2.

Binding Characteristics of Human Monoclonal Antibodies by EIA

ELISA analysis confirmed that purified antibody from the two hybridomas,C371A (also called Mab CNTO 95) and C372A, bind alphaVbeta in aconcentration-dependent manner. FIG. 1 shows the results of the relativebinding efficiency of the antibodies. Fifty percent binding is achievedat 0.07 and 0.7 μg/mL for C372A and CNTO 95 respectively. In the sameassay, c7E3 IgG demonstrated fifty-percent maximal binding at 0.07μg/mL.

Competition of Binding of Mab CNTO95 to M21 Cells by CommerciallyAvailable Anti-Integrin Mabs

By single-color analysis, none of the murine anti-alphaVbeta3,anti-alphaVbeta5, anti-beta3, or anti-alphaV antibodies competed withCNTO 95 for binding to M21 cells (Table 1). This experiment alsodemonstrates that CNTO 95 binds to M21 cells in a dose dependent manner.The two-color analysis demonstrated that the murine anti-alphaVbeta3,anti-alphaVbeta5, anti-beta3, or anti-alphaV antibodies were able tobind to M21 cells (data not shown).

TABLE 1 Competition of Binding of CNTO95 to M21 Cells by Murineanti-Integrin Mabs FITC-goat anti-human Fc-labeled CNTO95 2 μg/mL 20μg/mL Competing Antibody MCF % Positive MCF % Positive negative (noGenO95) 2.69 2.69 Positive (saline) 4.33 100% 14.33 100% m7E3 IgG 5.73132% 14.72 103% LM609 (anti-α_(v)β₃) 4.78 110% 13.34  93% anti-β₃(Chemicon) 5.42 125% 13.10  91% anti-β₃ (AMAC) 4.61 106% 13.10  91% P1F6(anti-α_(v)β₅) 4.87 112% 14.46 101% VNR139 (anti-α_(v)) 4.61 106% 14.86104% MCF = Median Channel Fluorescence

Inhibition of αVβ3 or αVβ5 Dependent M21 Cell or MDA-MB435-L2 CellAdhesion to Vitronectin Coated Plates by αVβ3/αVβ5 Specific Mabs

M21 cells adhere to vitronectin coated plates in an αVβ3 and αVβ5dependent manner. Therefore, blockade of both αVβ3 and αVβ5 is requiredto completely inhibit M21 cell adhesion to vitronectin coated plates.C372A did not inhibit M21 cell adhesion in the presence or absence ofP1F6, anti-αVβ5 ascites (FIG. 2). GenO95 (CNTO 95) completely inhibitedM21 cell adhesion to vitronectin coated plates both with and withoutanti-α_(v)β₅ (P1F6) ascites, indicating that the antibody blocks bothαVβ3 and αVβ5. As a control for the assay parameters, ReoPro (c7E3 Fab)which blocks αVβ3 (in addition to GPIIb/IIIa) was included. ReoPro aloneonly partially inhibited M21 cell adhesion, ReoPro in the presence ofanti-α_(v)β₅ (P1F6) ascites completely inhibited adhesion, whichdemonstrates that M21 cells bind to vitronectin through both α_(v)β₃ orα_(v)β₅ integrins. Data were normalized to percent of maximal M21 cellbinding in the absence of antagonist +/−anti-αVβ5 (P1F6) ascites. Forantagonist titration without P1F6, data were normalized to maximal M21cell binding in the absence of antagonist or P1F6. For antagonisttitration in the presence of P1F6, data were normalized to maximalbinding in the absence of antagonist but in the presence of P1F6. Datawere graphed as percent of maximal binding (no antibody) and non-linearregression performed using GraphPad Prism.

CNTO95 Mab also demonstrated the ability to completely inhibitMDAMB435L2 cell adhesion to vitronectin at a minimal concentration of1.5 μg/mL (FIG. 3). These data, in combination with the data indicatinginhibition of M21 cell adhesion, confirm the ability of GenO95 tofunctionally inhibit the αVβ3 and/or αVβ5 receptor interaction withvitronectin.

Determination of Ca⁺⁺ Dependence for Binding of Anti-HumanalphaVbeta3/alphaVbeta 5 Mabs to Their Ligands

It is known that the presence of the cation calcium is necessary for theMab c7E3 to bind alphaVbeta 3 and is not a requirement for binding ofMab LM609 to αVβ3 as demonstrated in FIGS. 4 c and 4 d respectively.This experiment was conducted to assess whether calcium dependence alsoapplies to the binding characteristics of CNTO 95 or C372 for alphaVbeta3 or alphaVbeta 5 integrins. An excess concentration of EDTA wasintroduced into the assay format to chelate the Ca present within thebinding pocket of the integrin subunits and therefore, binding wasassessed in the absence of the cation. It was found that CNTO 95 andC372 binding to alphaVbeta 3 is not dependent upon the presence of Ca(FIGS. 4 a, 4 b). The same is true for CNTO 95 binding to alphaVbeta 5but not so, however, for C372 binding to alphaVbeta 5 (FIGS. 4 e, 4 f)as binding appears to be increased in the presence of Ca.

Conclusion

The GenO fusion was performed utilizing splenocytes from a hybrid mousecontaining human variable and constant region antibody transgenes thatwas immunized with human αVβ3. Two totally human αVβ3 reactive IgGmonoclonal antibodies of the IgG1κ isotype were generated. These Mabswere further characterized and it was found that both bind αVβ3 and αVβ5integrins. The binding of the two Mabs was demonstrated to be calciumindependent to αVβ3 and calcium dependent to αVβ5 only for C372 binding.Moreover, one Mab, GenO95 (CNTO 95), is able to completely inhibit thebinding of αVβ3 and αVβ5 to the ligand vitronectin in cell based assays.This Mab may prove useful in anti-angiogenic and other cancer relatedapplications.

References

-   1. Taylor et al., International Immunology 6:579-591 (1993).-   2. Lonberg et al., Nature 368:856-859 (1994).-   3. Neuberger, Nature Biotechnology 14:826 (1996).-   4. Fishwild et al., Nature Biotechnology 14:845-851 (1996).-   5. Gastl et al., Oncology 54: 177-184 (1997).-   6. Eliceiri, et al., J. Clin. Invest. 103: 1227-1230 (1999).-   7. Friedlander et al., Science 270: 1500-1502 (1995).-   8. Wayner, et al., J. Cell Biol. 113: 919-929 (1991).

Example 3 Binding Affinities for Alpha-V Subunit Antibody

CNTO 95, (CNTO 95) as described in Example 2, is a human monoclonalantibody generated by immunizing (CBA/J×C57/BL6/J, GenPharmInternational) F2 hybrid mice with α_(v)β₃ integrin purified from humanplacenta. The antibody is composed of human variable and IgG1 kappaconstant regions and found to be reactive to both α_(v)β₃ and α_(v)β₅,suggesting a specificity for the alpha chain shared by both integrinmolecules.

The purpose of this study is to characterize the binding affinity ofGenO.05 for α_(v)β₃ and α_(v)β₅ purified integrins and for beta integrinexpressing cell lines. For further characterization, the binding valueswill be compared between CNTO 95 and ReoPro.

Abbreviations

K_(D), equilibrium dissociation constant, expressed in M

Bmax=maximal number of binding sites

Materials And Methods

Cell Lines

A375S2 cells, a human melanoma cell line expressing α_(v)β₃ and α_(v)β₅integrins, were cultured in Dulbelcco's minimal media (DMEM) containing10% fetal bovine serum (FBS, Cell Culture Labs), 1 mM sodium pyruvate,0.1 mM nonessential amino acids, and 2 mM L-glutamine (all from JRHBiosciences).

HT29 cells, a human colon carcinoma cell line expressing α_(v)β₅ andminimal α_(v)β₃ (NB 4546, p207) were cultured in DMEM containing 10%FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 2 mML-glutamine.

M21 cells, a human melanoma expressing α_(v)β₃ and α_(v)β₅ integrins,obtained from Dr. J. Jakubowski (Eli Lilly, Inc.), were cultured in RPMImedia (JRH Biosciences) containing 10% FBS, 1 mM sodium puruvate, 0.1 mMnonessential amino acids, and 2 mM L-glutamine.

Integrins

α_(v)β₃ lot JG22499 was purified at Centocor from human placenta.Another α_(v)β₃ integrin lot (octyl formulation, lot 19100991) waspurchased from Chemicon. α_(v)β₅ (Triton formulation, lot 20030055, lot1910990 and octyl formulation, lot 19060747) was purchased fromChemicon.

Antibodies

CNTO 95 was purified from cell culture supernatant by Protein Achromatography. ReoPro was manufactured at Centocor, Inc. LM609, amurine anti-human α_(v)β₃ antibody, (1976ZK, lot 20020559 and lot1910329) and P1F6, a murine anti-human α_(v)β₅ antibody (1961 P-K, lot17110560) were purchased from Chemicon.

Radiolabeling

Antibodies were radiolabeled with 125-I Na (Amersham, Ill.) usingIodobeads (Pierce Chemicals, Ill.) to a specific activity of 1-2 μCi/μg.Antibody concentration (mg/ml) was determined by dividing the adsorption(OD/ml) at 280 nm by 1.4. Specific activity of the iodinated antibodywas determined by diluting the antibody and counting an aliquot in thegamma counter or Topcounter (Packard).Specific activity(cpm/ug)=cpm/volume(ml)×dilution factorconcentration(μg/ml determined by OD₂₈₀reading)

Integrin-Coated Plate Binding Assay

α_(v)β₃ or α_(v)β₅ integrin was diluted to 1 μg/ml in Tris-bufferedsaline (TBS, 10 mM Tris, 100 mM NaCl, pH 7.5) containing 2 mM calciumchloride (TBS/Ca⁺⁺) and coated at 50 μl per well onto 96 wellpolystyrene Linbro plates (Flow/ICN) overnight at 4° C. Plates werewashed with TBS/Ca⁺⁺ and blocked with 1% bovine serum albumin (BSA) inTBS/Ca⁺⁺ for 1 h at room temperature. Fifty microliters of dilutedantibody was added in triplicate to coated wells and incubated for 2 hat 37° C. After three washes with TBS-Tween buffer (TBS+0.1% Tween 20),peroxidase conjugated goat anti-human IgG F(ab′)₂ (H+L, Jackson lot16869), at 1:40:000 dilution in 1% BSA-TBS was added and incubated for 1h at room temperature. Plates were washed three times, and developedwith o-phenylenediamine dihydrochloride substrate solution (OPD, Sigma)consisting of 0.1 M citric acid, 0.2M sodium phosphate, 0.01% H₂O₂ and 1mg/ml OPD. Color development was stopped after 15 min at roomtemperature with 0.3 N H₂SO₄, and plates were read at OD₄₉₀ nm in theMolecular Dynamics plate reader.

Binding curves were generated with GraphPad PRISM (version 3, GraphPadSoftware). Results were expressed as % maximal binding of the saturationvalue. K_(D), the equilibrium dissociation binding constant (expressedas M), was determined from a non-linear regression fit of the data usingPRISM.

Cell Binding Assay

Fifty microliters of diluted radiolabeled antibody in 2% RPMI mediacontaining 2% bovine serum albumin (JRH Biosciences) were added intriplicate to confluent cells cultured in 96 well tissue culture plates(Packard). Cells were incubated for 1.5 h at 37° C.; gently washed threetimes with Hanks buffered saline containing calcium and magnesium(HBSS++, JRH Biosciences) and then aspirated. One hundred microliters ofMycosinct 20 (Packard) was added per well, and cell-bound radioactivitywas quantified in the TopCounter (Packard).

To determine nonspecific binding, experiments were performed with asimilar set of dilutions in the presence of 100-fold excess of unlabeledantibody.

To determine the number of cells plated in each well, cells from severalwells were removed with trypsin, pooled and counted under themicroscope. The receptor number per cell was calculated as follows:Receptor number/cell=specific bound cpms×6.023×10²³molecules/molespecific activity(cpm/g)×mol.wt. (g/mole)×cell number

Bmax, the maximal binding sites per cell, and the K_(D) were determinedfrom a nonlinear regression fit of the data using PRISM.

Results and Discussion

Determination of the binding affinity values was performed by measuringthe binding of various concentrations of CNTO 95 (and ReoPro) topurified α_(v)β₃ and α_(v)β₅ integrins and to cell surface receptors atequilibrium. The saturation binding curves were rectangular hyperbolas,suggesting a single receptor binding site for CNTO 95 and ReoPro (FIGS.5-6; Motulsky H, 1999). Analysis of these saturation binding data(sometimes called Scatchard experiments) were performed using a one-sitehyperbola nonlinear regression fit in PRISM to obtain an affinity,K_(D), and receptor number, Bmax (Motulsky H, 1999).

Several lots of CNTO 95, ReoPro and purified integrins were used toensure an accurate determination of binding affinity values. Thesaturation binding curve of CNTO 95 on an α_(v)β₃ coated plate (FIG. 5A)and the binding curve of ReoPro on an α_(v)β₃ coated plate (FIG. 5B)represent the mean and standard deviation of six separate experiments.Results obtained with Triton formulation of α_(v)β₃ were found to bemore reproducible than those obtained from the octyl formulation. Onα_(v)β coated plates, the CNTO 95 mean K_(D) was 2.1±1.33×10⁻¹⁰ M; andthe mean ReoPro Kd was 2.5±1.46×10⁻¹⁰ M.

The saturation binding curve of CNTO 95 on an α_(v)β₅ coated plate (FIG.6A) and the binding curve of ReoPro on an α_(v)β₅ coated plate (FIG. 6B)are shown as the mean and standard deviation of six separateexperiments. Results obtained with the octyl formulation were moreconsistent than those obtained with the Triton formulation. The CNTO 95mean K_(D) on α_(v)β₅ was 2.5±1.04×10⁻¹¹ M. ReoPro showed no binding andno dose-response on α_(v)β₅ coated plates.

The binding affinity values for purified integrins were compared tobinding to receptors expressed on various cell lines. FIG. 7A-C showsthe binding of 125-I CNTO 95 with A375S2 cells which express α_(v)β₃ andα_(v)β₅ (FIG. 7A). Mean affinity values on A375S2 cells were:Kd=5.2±2.04×10⁻⁹ M; and 120,000±37,000 receptors/cell. HT-29 cellsexpress α_(v)β₅. Affinity values for 125-I CNTO 95 binding to HT-29cells were: Kd=1.3±3.76×10⁻¹⁰ M; and 81,000±24,000 receptors/cell (FIG.7B). M21 cells express α_(v)β₃ and α_(v)β₅ integrins. 125-I CNTO 95binding to M21 cells were: Kd=8.5±3.03×10⁻⁹ M; and 200,000±80,000receptors/cell (FIG. 7C).

Similar cell binding studies were performed with 125-I ReoPro on variouscell lines. FIG. 8A-C shows the binding of 125-I ReoPro with A375S2cells and the mean values obtained were: Kd=22±3.7×10⁻⁹ M; and370,000±190,000 receptors/cell (FIG. 8A). On HT-29 cells, 125-I ReoProshowed minimal binding (FIG. 8B). 125-I ReoPro binding to M21 cellsshowed: Kd=10±2.00×10⁻⁹ M and 660,000±120,000 receptors/cell (FIG. 8C).The binding values of 125-I ReoPro on M21 cells are consistent withvalues previously published (Tam et al, 1998).

A summary of binding results is shown in Tables 2-3.

TABLE 2 Summary of CNTO 95 and abciximab affinities to purifiedintegrins alphaVbeta 3 coated plate alphaVbeta 5 coated plate mAb (n =6) Kd (M) (n = 6) Kd (M) CNTO 95 2.1 + 1.33 × 10⁻¹⁰ 2.5 + 1.04 × 10⁻¹⁰abciximab 2.5 + 1.46 × 10⁻¹⁰ Negligible

TABLE 3 Summary of CNTO 95 and abciximab affinities to cells A375S2cells HT-29 cells M21 cells A375S2 cells Receptors HT-29 cells ReceptorsM21 cells Receptors Kd (M) per cell Kd(M) per cell Kd (M) Per cell CNTO95 5.2 ± 2.04 × 10⁻⁹ 120,000 ± 37,000  1.3 ± 0.38 × 10⁻⁹ 81,000 ± 24,0008.5 ± 3.03 × 10⁻⁹ 200,000 ± 80,000  (n = 5) (n = 7) (n = 5) (n = 7) (n =4) (n = 8) abciximab  22 ± 3.7 × 10⁻⁹ 370,000 ± 190,000 NegligibleNegligible  10 ± 2.00 × 10⁻⁹ 660,000 ± 120,000 (n = 3) (n = 6) (n = 4)(n = 4) (n = 3) (n = 7) anti α_(v)β₃ nd 300,000 nd nd nd nd LM609 (n =2) anti-α_(v)β₅ nd 70,000 ± 50,000 nd 73,000 nd 44,000 P1F6 (n = 4) (n= 1) (n = 2)

Several observations were notable in the binding characterizations.Affinity values (Kd) of CNTO 95 on α_(v)β₅ were lower than on α_(v)β₃.Lower Kd values indicate a higher affinity; thus the affinity for CNTO95 binding to α_(v)β₅ purified integrin was about 8-fold higher thanbinding to α_(v)β₃ purified integrin. However, when both integrinreceptors are present on the same cells, the overall affinity value moreclosely approximates the value corresponding to the integrin in greaterabundance. Thus, on A375S2 and M21 cells where there is more α_(v)β₃than α_(v)β₅, the affinity of CNTO 95 binding to these cells was similarto the affinity on α_(v)β₃, ˜7×10⁻⁹ M. In contrast, on HT-29 cells whichexpress α_(v)β₅, the CNTO 95 affinity was slightly higher, 1×10⁻⁹ M. Theapproximately 2-fold discrepancy in receptor sites per cell between CNTO95 and ReoPro binding may be explained by the difference in antibodyvalency. CNTO 95 (IgG) is bivalent and likely binds two adjacentreceptors, whereas ReoPro (Fab) is monovalent and can only bind to onereceptor (BRD930001).

References

-   Fraker D J, Speck J C. Protein and cell membrane iodination with a    sparingly soluble chloramide    1,3,4,5-tetrachloro-3a-diphenyl-glycoluryl. Biochem Biophys Res    Commun. 80:849, 1978.-   Motulsky H. Analyzing Data with GraphPad Prism. GraphPad Software,    Inc. San Diego, Calif. 1999.-   Tam S H, Sassoli P M, R Jordan, M T Nakada. Circulation, 1999.

Example 4 Effect of Alpha-V Subunit Antibody on Angiogenesis Modulation

GenO95 is a human IgG1κ monoclonal antibody that recognizes integrinsαvβ3 and αvβ5 as well as α_(v)β₁ and α_(v)β₆. These integrinsparticipate in endothelial cell adhesion, migration, survival andproliferation, processes that are important for angiogenesis.Endothelial cell sprouting mimics angiogenesis in vitro because itinvolves cell adhesion, migration, proliferation and survival. Weutilized the sprouting assay to determine whether GenO95 could inhibitαvβ3 and αvβ5 function. This example describes that GenO95 is aninhibitor of sprouting of endothelial cells that are cultured in threedimensional fibrin matrix, thereby demonstrating that this antibody mayhave potential anti-angiogenic properties.

There is now considerable evidence that progressive tumor growth isdependent upon angiogenesis, the formation of new blood vessels. Theseblood vessels provide tumors with nutrients and oxygen, carry away wasteproducts and act as conduits for the metastasis of tumor cells todistant sites (1). Recent studies have further defined various roles ofintegrins in the angiogenic process. Integrins are subunitictransmembrane proteins that play an important role in mediating celladhesion, migration, survival, and proliferation (2). Expression ofintegrin αvβ3 is minimal on resting or normal blood vessels but issignificantly up-regulated on angiogenic vascular cells (1-3). Theclosely related but distinct integrin αvβ5 has also been shown tomediate the angiogenic process. An antibody generated against αvβ3blocked basic fibroblast growth factor (bFGF) induced angiogenesis,whereas an antibody specific to αvβ5 inhibited vascular endothelialgrowth factor (VEGF) induced angiogenesis (1-5).

Angiogenesis can be mimicked in vitro by an endothelial sprouting assay.This system involves endothelial cell migration and proliferation.GenO95 is a human monoclonal antibody that recognizes integrins αvβ3 andαvβ5, and these integrins regulate endothelial cell migration andproliferation. Therefore, we determined whether GenO95 could inhibitsprouting of endothelial cells. This example describes experiments thatdemonstrate that GenO95 inhibits sprouting of human endothelial cellsgrowing in a fibrin matrix.

Materials

Human basic fibroblast growth factor (bFGF) and human vascularendothelial growth factor 165 (VEGF₁₆₅) were obtained from R&D Systems(Minneapolis, Minn.). MAB 1976Z (LM609), a monoclonal antibody againstintegrin αvβ3 and MAB1961 (PIF6), a monoclonal antibody against integrinαvβ5 were purchased from Chemicon (Temecula, Calif.). ReoPro and GenO95were obtained from Centocor's Clinical Pharmacology and AntibodyTechnology Department. Human fibrinogen (plasminogen free, >95%clottable protein) and bovine skin gelatin were purchased from Sigma(Saint Louis, Mich.).

Cell Lines

Huvecs, Human umbilical vein endothelial cells, were purchased fromClonetics (Walkersville, Mass.). Huvecs were cultured in endothelialbasal media (EBM) kit (Clonetics) containing 10% FBS, long Rinsulin-like growth factor-1, ascorbic acid, hydrocortisone, humanepidermal growth factor, human vascular endothelial growth factor,hFGF-b, gentamicin sulfate, and amphotericin-B. Cells were incubated at37° C. and 5% CO₂ and media was changed every 2 to 3 days. Only passages3 to 8 were used in all experiments.

Fibrin Microcarrier-Based Sprouting Assay

A modification of the methods of Nehls and Drenckhahn (6) was used tomeasure capillary tube formation in three-dimensional fibrin-basedmatrix. Gelatin-coated cytodex-3 microcarriers (MCs, Sigma) wereprepared according to recommendations of the supplier. Freshlyautoclaved MCs were suspended in EBM-2+20% FBS and endothelial cellswere added to a final concentration of 40 cells/MC. The cells wereallowed to attach to the MCs during a 4-hour incubation at 37° C. TheMCs were then suspended in a large volume of medium and cultured for 2to 4 days at 37° C. in 5% CO₂ atmosphere. MCs were occasionally agitatedto prevent aggregation of cell coated beads. MCs were embedded in afibrin gel that was prepared as follows: human fibrinogen (2 mg/ml) wasdissolved in plain, bFGF or serum containing EBM-2 media. This solutionalso contained various antibodies. To prevent excess fibrinolysis byfibrin-embedded cells, aprotinin was added to the fibrinogen solutionand to growth media at 200 U/ml. Cell-coated microcarriers were added tothe fibrinogen solution at a density of 100 to 200 MCs/ml (50-100beads/per well-48 well plate) and clotting was induced by addition ofthrombin (0.5 U/ml). After clotting was complete, 0.5 ml solution(containing all components described above except fibrinogen andthrombin) was added to the fibrin matrices. The plates were incubated at37° C. and 5% CO₂ for 1 to 3 days. After 1-3 days, gels were fixed with3% paraformaldehyde dissolved in PBS, and the number of capillarysprouts with length exceeding the diameter of the MC bead (150 μm) wasquantified.

Results and Discussion

Huvecs can form capillary-like sprouts when cultured in a fibrin gel(FIG. 9). Endothelial cells migrate outwards from the gelatin coatedbeads and extend into long filopodia. The long sprouts consist ofseveral cells forming a lumen. This process resembles microcapillaryformation in vivo, because it involves endothelial cell migration,invasion and cell proliferation. Quantification of sprout formationrevealed that GenO95 inhibited endothelial cell sprout formation in bFGFor complete media (FIG. 10). Combination of LM609 and P1F6 routinelyinhibited sprouting more effectively than GenO95 (FIG. 11).

Conclusion

Formation of new blood vessels from existing blood vessels is a hallmarkof angiogenesis. This process can be mimicked in vitro by theendothelial sprouting assay. These sprouts represent microcapillariesthat are formed in response to angiogenic stimuli such as bFGF or avariety of stimuli that are present in serum. GenO95 dose dependentlyinhibited bFGF- and complete media-stimulated endothelial cellsprouting, suggesting that this antibody can effectively inhibit αvβ3and αvβ5 function. Why GenO95 was not as effective as the combination ofLM609 and P1F6 is unknown, but it is possible that GenO95 recognizesαvβ3 and αvβ5 with lower affinity when compared to LM609 and P1F6,respectively. Collectively, these data demonstrate that GenO95 caninhibit the complex process of microcapillary formation in vitro.

References

-   1. Gastl G, Hermann T, Steurer M, Zmija J, Gunsilius E, Unger C, and    Kraft A. 1997. Angiogenesis as a Target for Tumor Treatment.    Oncology 54:177-184.-   2. Eliceiri B P, and Cheresh D A. 1999. The role of αV integrins    during angiogenesis: insights into potential mechanisms of action    and clinical development. The Journal of Clinical Investigation    103:1227-1230.-   3. Brooks P C, Montgomery A M, Rosenfeld M, Reisfeld R A, 1994.    Integrin αvβ3 antagonists promote tumor regression by inducing    apoptosis of angiogenic blood vessels. Cell 79: 1157-1164.-   4. Enenstein J, Walweh N S, and Kramer R H. 1992. Basic FGF and    TGF-β differentially modulate integrin expression of human    microvascular endothelial cells. Exp. Cell Res. 203:499-503.-   5. Friedlander M, Brooks P C, Shaffer R W, Kincaid C M, Varner J A,    and Cheresh D A. 1995. Definition of two angiogenic pathways by    distinct αV integrins. Science 270:1500-1502.-   6. Nehls, V and Drenckhahn, D. 1995. A novel, microcarrier-based in    vitro assay for rapid and reliable quantification of    three-dimensional cell migration and angiogenesis. Microvascular    Res. 50:311-322.

Example 5 Effect of Alpha-V Subunit Antibody on Endothelial and TumorCell Adhesion, Migration and Invasion

(CBA/J×C57/BL6/J) F₂ hybrid mice (1-4) containing human variable andconstant region antibody transgenes for both heavy and light chains wereimmunized with human placental αVβ3. One fusion yielded a totally humanαVβ3 reactive IgG1κ monoclonal antibody named GenO95. The totally humanantibody was found to be reactive to the αVβ3 and αVβ5 integrins (5).These integrins participate in endothelial and tumor cell adhesion,migration, and invasion. Therefore, we characterized the effect of CNTO95 on integrin mediated cell motility. CNTO 95 inhibits human umbilicalvein endothelial (HUVEC) and human melanoma cell binding to vitronectin,denatured collagen, fibrinogen and fibrin, but it does not block celladhesion to fibronectin and type I collagen. GenO95 also inhibitsmigration of endothelial cells that have been stimulated with basicfibroblast growth factor and low-dose serum. GenO95 inhibits invasion oftumor cells through a fibrin gel. In conclusion, GenO95 functionallyblocks αVβ3 and αVβ5 in a variety of cell-based assays in vitro.

Abbreviations

-   -   BSA—bovine serum albumin    -   CO₂—carbon dioxide    -   DMSO—dimethyl sulfoxide    -   FBS—fetal bovine serum    -   Ig—immunoglobulin    -   Mab—monoclonal antibody    -   OD—optical density    -   RT—room temperature    -   HUVECS—human umbilical vein endothelial cells    -   bFGF—bovine basic fibroblast growth factor

Introduction

There is now considerable evidence that progressive tumor growth isdependent upon angiogenesis. The formation of new blood vessels providetumors with nutrients and oxygen, carry away waste products and act asconduits for the spread of tumor cells to distant sites. Several studieshave defined the role of integrins in the angiogenic process. Integrinsare subunitic trans-membrane proteins that play a critical role in celladhesion to the extracellular matrix (ECM) and mediate cell survival,proliferation and migration (6). During the angiogenic process, αvβ3 andαvβ5 are upregulated on the surface of activated endothelial cells,which in turn helps these cells to migrate and proliferate (6). Anantibody generated against αVβ3 blocks basic fibroblast growth factor(bFGF) induced angiogenesis, whereas an antibody specific to αVβ5inhibits vascular endothelial growth factor (VEGF) induced angiogenesis(6, 7). In addition to regulating angiogenesis, αVβ5 and αVβ3 regulatetumor cell adhesion, migration and invasion, processes required fortumor cell metastases. Previous studies indicated that CNTO 95 binds topurified αVβ5 and αVβ3 integrins, therefore, we determined whether thisantibody could functionally block αVβ3- and αVβ5-mediated endothelialand tumor cell adhesion, migration and invasion.

Materials and Methods

Materials

Bovine fibroblast growth factor (bFGF) and human vascular endothelialgrowth factor 165 (VEGF₁₆₅) were obtained from R&D Systems (Minneapolis,Minn.). MAB 1976Z (LM609), a monoclonal antibody against integrin αvβ3and MAB1961 (PIF6), a monoclonal antibody against integrin αvβ5 werepurchased from Chemicon (Temecula, Calif.). ReoPro (lot: 94A04ZE) andCNTO 95 (lot: JG100899) were obtained from Centocor. BIOCOAT cellculture inserts (pore size: 8 μm) were purchased from Becton Dickinson(Bedford, Mass.). Vybrant™ cell adhesion assay kit (V-13181) waspurchased from Molecular Probes (Eugene, Oreg.). Human plasminogen freefibrinogen (VWF/Fn depleted) was purchased from Enzyme Research Labs(South Bend, Ind.). Bovine skin gelatin was purchased from Sigma (SaintLouis, Mo.). Human vitronectin was purchased from Promega (Madison,Wis.), and type I collagen was purchased from GIBCO BRL (Gaithersburg,Md.).

Cell Lines

Human umbilical vein endothelial cells (HUVECS), were purchased fromClonetics (Walkersville, Mass.), and they were cultured in EBM mediumkit (Clonetics) containing 10% FBS, long R insulin-like growth factor-1,ascorbic acid, hydrocortisone, human epidermal growth factor, humanvascular endothelial growth factor, gentamicin sulfate andamphotericin-B. Cells were grown at 37° C. and 5% CO₂ and media waschanged every 2 to 3 days. Cells were passaged when they reached 80%confluence. Passages 3 to 8 were used in all experiments.

The A375S.2 human melanoma cell line expressing the αVβ3 and αVβ5integrins was obtained from Centocor Cell Bank where the cell line wasdeemed free of mycoplasma and bacterial contaminants. The cells werecultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1mM sodium pyruvate, and 0.1 mM non-essential amino acids.

Human colon carcinoma HT29 cells were obtained from Centocor CellBiology Service Department, where the cell line was deemed free ofmycoplasma and bacterial contaminant. The cells were cultured in α-MEMmedium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodiumpyruvate, and 0.1 mM nonessential amino acids.

Flow Cytometry

For the detection of surface integrins, cells were harvested, rinsed,suspended in unsupplemented RPMI media, and sequentially incubated for60 minutes on ice with anti-integrin mAb (10 μg/ml) and FITC-labeledgoat anti-mouse antibody (1:100) or FITC-labeled anti-integrin antibody(10 μg/ml). Absence of primary antibody or substitution of primaryantibody with isotype matched antibody served as negative controls.Cells were immediately analyzed with a FACS Scan II flow cytometer(Becton Dickinson, Mountain View, Calif.).

Adhesion Assay

Microtiter plates (Linbro-Titertek, ICN Biomedicals, Inc) were coated at4° C. overnight with vitronectin (1 μg/ml), gelatin (0.1%), fibrinogen(100 μg/ml), type I collagen (10 μg/ml), or fibronectin (10 μg/ml).Immediately before use plates were rinsed with PBS and blocked for 1hour with 1% BSA/PBS (pH 7.4). Fibrin-coated Microtiter wells wereformed by thrombin treatment (1 U/ml) of fibrinogen. Adherent cells(HUVECS HT29 and A375S.2) were labeled with Calcein AM fluorescent dye(Molecular Probes, Eugene, Oreg.) according to the manufacturer'sinstructions, harvested, washed twice, and suspended in 0.1% BSA in DMEMmedium. After cell density was adjusted to 5×10⁵/ml, cells wereincubated with various concentrations of antibodies for 15 min at 37° C.The cell-antibody mixture was added to wells (100 μl per well) andincubated for 1 h at 37° C. Plates were rinsed twice with PBS to removeunbound cells and adhesion was measured in a fluorescence plate reader(Fluoroskan) at 485-538 nm. Cell adhesion to BSA-coated wells served asa negative control. Isotype matched antibodies served as a negativecontrol.

Chemotactic Migration Assay

Cell migration assays were performed in 24-Transwell chambers with apolystyrene membrane (6.5 mm diameter, 10 μm thickness, and a pore sizeof 8 μm). Sub-confluent 24-hr cell cultures (HUVECS or A375S.2) wereharvested with trypsin-EDTA, washed twice, and resuspended in theirrespective serum free medium containing 0.1% BSA. Cells (100,000/500 μl)were added to the upper chamber in the presence or absence ofantibodies. To facilitate chemotactic cell migration, 750 μl of mediumcontaining 0.1% BSA and vitronectin (2 μg/ml) or serum (2% for HUVECSand 10% for A375S2 cells) was added to the bottom chambers and the platewas placed in a tissue culture incubator. Migration was terminated after4 to 8 hrs by removing the cells on the top with a cotton swab and thenthe filters were fixed with 3% paraformaldehyde and stained with CrystalViolet. The extent of cell migration was determined by light microscopyand images were analyzed using the Phase 3 image analysis software (GlenMills, Pa.). The software analyzes the total area occupied by thestained cells on the bottom side of the filter and this is directlyproportional to the extent of cell migration.

Haptotactic Migration Assay

Cell migration assays were performed using the transwell chambers asdescribed above with slight modifications. Briefly, the underside of themembrane was coated with vitronectin (2 μg/ml) for 60 minutes at roomtemperature, and then blocked with a solution of 1% BSA/PBS at roomtemperature for 60 min. Next, membranes were washed with PBS and airdried. Serum free medium (750 μl) containing 0.1% BSA and bFGF (20ng/ml) was added to the lower chambers. Sub-confluent 24 h cultures wereharvested with trypsin-EDTA, washed twice, and resuspended in serum freemedium. Cells (100,000/500 μl) were added to the upper chambers in thepresence or absence of antibodies. The chambers were placed in a tissueculture incubator and migration was allowed to proceed for 6 h. Extentof cell migration was determined as described above.

Invasion Assay

Fibrinogen (Plasminogen-free, 100 μl of 10 mg/ml) and 100 μl of 1 U/mlthrombin was mixed and immediately added to the top chamber of 24 welltranswell plates (6.5 mm diameter, 10 μm thickness and a pore size of8.0 μm, Costar). The plates were incubated at 37° C. for 30 minutes toform a fibrin gel. Confluent tumor cells (A375S.2) were trypsinized,centrifuged, resuspended in basal medium supplemented with 0.1% BSA and10 μg/ml plasminogen

(Enzyme Research Labs, South Bend, Ind.) with various concentrations ofantibodies, and incubated for 15 minutes at room temperature. Cells(100,000/500 μl) were added to the upper chamber in the presence orabsence of antibodies. The lower compartment of the invasion chamber wasfilled with 0.75 ml of 10% FBS-DMEM, which served as a chemoattractantand the plate was transferred to a tissue culture incubator. After 24hours, invasion was terminated by removing the cells on the top with acotton swab, and the filters were fixed with 3% paraformaldehyde andstained with Crystal Violet. The extent of cell migration was analyzedusing the Phase 3 image analysis software as described above.

Results and Discussion

CNTO 95 inhibits αvβ3- and αvβ5-mediated cell adhesion

Since CNTO 95 binds to αVβ3 and αVβ5 integrins, we determined whetherour tumor cells (A375S.2 and HT29) and endothelial cells express theseintegrins. Flow cytometry indicated that A375S.2 and HUVEC cells expressboth αVβ3 and αVβ5 integrins, but HT29 cells express αVβ5, but notα_(v)β₃ integrin (FIG. 12A-I).

HT29 cells (12A, B and C) express αvβ5, but not αvβ3 integrin on theirsurface. HUVEC (12D, E and F) and A375S.2 (12G, H and I) cells expressαvβ5 and αvβ3 integrin on their surface. Tumor cells and endothelialcells were stained by immunofluorescence and analyzed by flow cytometry.The histogram on the left represents background fluorescence in thepresence of isotype matched antibody. The histogram on the rightindicates positive staining. A, D, G, LM609 (mAb directed to αvβ3, 10μg/ml); B, E, H, PIF6 (mAb directed to αvβ5, 10 μg/ml); and C, F, I,GenO95 (10 μg/ml).

The effect of CNTO 95 on adhesion of HUVEC, A375S.2 and HT 29 cells tovarious matrix proteins was determined in detail. GenO95 completelyinhibited adhesion of HUVEC and A375S.2 cells to vitronectin, andpartially to fibrinogen, gelatin and fibrin coated plates, indicatingthat the antibody can block αVβ3 and αVβ5 (FIGS. 13 and 14, Table 1 and2). GenO95 completely inhibited HT-29 cell adhesion to vitronectincoated plates, indicating that the antibody blocks αVβ5 (FIG. 15).GenO95 completely inhibited adhesion of HUVEC and A375S.2 cells tovitronectin coated plates, indicating that the antibody blocks αVβ3 andαVβ5 (FIGS. 13 and 14). Data were graphed as percent of maximal binding(no antibody) and non-linear regression performed using GraphPad Prism.

Adhesion of HUVECS to matrix protein-coated plates. Adhesion assay wasperformed as described in Methods. Plate was read on a fluorometer at485-538 nm. Cell adhesion to BSA coated wells served as a negativecontrol. In FIG. 13, the extent of cell adhesion in the presence ofvarious concentrations of antibody was plotted as a percent of celladhesion in the absence of antibody that was considered as 100%. Eachdata point is the mean of triplicate determinations (+/−SD).

Adhesion of human melanoma cells to matrix protein-coated plates.Adhesion assay was performed as described in Methods. Cell adhesion toBSA coated wells served as a negative control. In FIG. 14 the extent ofcell adhesion in the presence of various concentrations of antibody wasplotted as a percent of cell adhesion in the absence of antibody thatwas considered as 100%. Each data point is the mean of triplicatedeterminations (+/−SD).

Table 4 shows the extent of HUVECs adhesion to vitronectin, gelatin,fibrinogen, fibrin, fibronectin and type I collagen in the presence ofvarious concentration of antibody was plotted as a percent of celladhesion in the absence of antibody that was considered as 100%. Eachdata point is the mean of triplicate determinations (+/−SD). Theconcentration of antibodies used was 10 μg/ml.

TABLE 4 Type I Vitronectin Gelatin Fibrinogen Fibrin Fibronectincollagen Human IgG  96.3 ± 11.4 109.0 ± 8.8  108.0 ± 6.3  99.7 ± 4.5 96.8 ± 4.7 99.3 ± 4.1 LM609 26.3 ± 3.7 36.5 ± 4.7 14.3 ± 2.5 48.1 ± 1.5102.8 ± 7.2 108.8 ± 12.7 PIF6 39.8 ± 5.9  94.4 ± 15.1 94.5 ± 4.2 96.7 ±4.5 103.2 ± 3.8 115.7 ± 8.1  LM609-  3.7 ± 0.4 32.2 ± 5.2 10.7 ± 1.130.7 ± 8.9  99.6 ± 4.7 116.2 ± 4.1  PIF6 CNTO 95  3.3 ± 0.6 54.8 ± 4.034.5 ± 1.7 45.1 ± 2.4 101.6 ± 6.1 97.7 ± 3.9 ReoPro 54.9 ± 0.9  2.5 ±2.3  8.7 ± 2.9 35.8 ± 3.0  96.3 ± 2.8 99.6 ± 6.0

Table 5 shows the extent of A375S.2 cells adhesion to vitronectin,gelatin, fibrinogen, fibrin, fibronectin and type I collagen in thepresence of various concentration of antibody. The data is expressed asa percent of cell adhesion in the absence of antibody that wasconsidered as 100%. Each data point is the mean of triplicatedeterminations (+/−SD). The concentration of antibodies used is 10μg/ml.

TABLE 5 Type I Vitronectin Gelatin Fibrinogen Fibrin Fibronectincollagen Human IgG 104.0 ± 5.3   94.6 ± 12.4 102.5 ± 5.9  99.5 ± 4.0100.0 ± 5.5  99.1 ± 3.3 LM609 42.1 ± 6.1 25.2 ± 7.1 14.0 ± 1.8 50.0 ±1.9 104.0 ± 8.1 100.0 ± 1.5 PIF6 28.5 ± 3.8 87.4 ± 7.8 99.4 ± 3.6 92.9 ±4.7 101.0 ± 5.7 101.0 ± 7.3 LM609-  0.9 ± 0.3  1.1 ± 1.5 10.3 ± 2.6 47.6± 3.2 109.0 ± 4.1 102.0 ± 4.6 PIF6 CNTO 95  1.4 ± 0.4 23.2 ± 7.2 11.4 ±2.8 43.3 ± 3.5 103.0 ± 4.5 104.0 ± 5.9 ReoPro 38.1 ± 0.7  6.0 ± 1.0  6.5± 2.1 12.9 ± 3.8 104.0 ± 5.6  93.1 ± 3.1

The adhesion human colon carcinoma HT29 cells to vitronectin in thepresence of antibody was performed as described above. Cell adhesion toBSA coated wells served as a negative control. The data shown in FIG. 15are plotted as percent of maximum binding (absence of antibody), and arethe mean of triplicate determinations (+/−SD).

CNTO95 Blocks Human Melanoma and Endothelial Cell Migration

Integrins αVβ3 and αVβ5 participate in cell migration, therefore wedetermined whether CNTO 95 could block vitronectin-stimulated cellmigration. Vitronectin-stimulated cell migration involves αVβ3 and αVβ5.CNTO 95 dose dependently inhibited endothelial cell migration whenvitronectin was used as a chemoattractant (FIG. 17). Interestingly, CNTO95 also inhibited migration of both HUVECS and A375S.2 cells to serum(FIGS. 18 and 19). These findings could be potentially important forangiogenic and tumor therapy because they suggest that the targets forCNTO 95, αVβ3 and αVβ5, are central receptors that are activated by avariety of migratory factors that are present in serum.

FIG. 16 shows the migration of HUVECS toward 2 μg/ml vitronectin. Theassay was performed as described in Methods and cells were allowed tomigrate for 6 h. Photomicrographs are representative fields (10×objective lens) of cell migration in FIG. 16A, absence of antibody,(16B), CNTO 95 (5 μg/ml), (16C), CNTO 95 (40 μg/ml). FIG. 16D isgraphical representation of cell migration in the presence of varyingconcentrations of GenO95. The data were normalized to percent of control(no antibody) which was considered as 100%, and each point is the meanof three transwell filters (+/−SD).

FIG. 17 shows the migration of HUVECS toward 2 μg/ml vitronectin in thepresence of antibodies to αvβ3 and αvβ5. The migration assay wasperformed as described in Methods, and cells were allowed to migrate for6 hours. LM609 and P1F6 are mAbs directed to αvβ3 and αvβ5,respectively. The data shown in FIG. 17 were normalized to percent ofcontrol (no antibody) which was considered as 100%, and each bar is themean of three transwell filters (+/−SD). BSA, mouse IgG and human IgGserved as negative controls. LM609-PIF6 represents combinations of bothantibodies. The antibodies and BSA were used at a concentration of 10μg/ml.

FIG. 18 shows the migration of HUVECS towards 2% FBS. Migration assaywas allowed to proceed for 4 h and the data was captured as described inMethods. FIG. 18(A) is a graphical representation of cell migration inthe presence of LM609, P1F6, combination of LM609+P1F6, isotype matchedcontrol antibodies (human and mouse). The antibodies and proteins wereused at a concentration of 10 μg/ml. FIG. 18(B) is a graphicalrepresentation of cell migration in the presence of ReoPro and GenO95.Photomicrographs are representative fields (10× objective lens) of cellmigration in FIG. 18(C), the absence of antibody, FIG. 18(D), GenO95 (5μg/ml), and FIG. 18(E), GenO95 (20 μg/ml). The data were normalized topercent of control (no antibody) which was considered as 100%, and eachpoint is the mean of three transwell filters (+/−SD).

FIG. 19 shows the migration of A375S.2 cells toward 10% FBS. Migrationassay was allowed to proceed for 4 h and the data was captured asdescribed in Methods. Antibodies were used at a concentration of 10μg/ml. FIG. 19(A) is a graphical representation of cell migration in thepresence of varying concentrations of GenO95. FIG. 19(B) is a graphicalrepresentation of cell migration in the presence of LM609, P1F6,combination of LM609+P1F6, isotype matched control antibodies (human andmouse). The data were normalized to percent of control, which wasconsidered as 100%, and each point is the mean of three transwellfilters (+/−SD). Photomicro-graphs are representative fields (10×objective lens) of cell migration in FIG. 19(C), absence of antibody,FIG. 19(D), GenO95 (5 μg/ml), and FIG. 19(E), GenO95 (20 μg/ml).

Results described above indicate that CNTO 95 blocks tumor andendothelial migration to vitronectin and serum. Next, we determinedwhether this antibody could inhibit bFGF-stimulated cell migration. Asshown in FIG. 20, bFGF stimulated HUVEC cell migration towardsvitronectin, and CNTO 95 significantly blocked this stimulated cellmigration.

FIG. 20 shows the migration of HUVECS towards vitronectin in thepresence of bFGF. The undersides of migration chamber filters werecoated with 2 μg/ml vitronectin, and the assay was performed asdescribed in Methods. Cells were allowed to migrate for 6 h. In FIG.20A-E, each data point is the mean of 3 transwell filters (+/−SD). FIG.20(A), bFGF; FIG. 20(B), CNTO 95 (5 μg/ml); FIG. 20(C), CNTO 95 (40μg/ml); FIG. 20(D), no-bFGF. FIG. 20(E), Inhibition of cell migration inthe presence of various antibodies is shown graphically.

GenO95 Blocks Human Melanoma Cell Invasion

Results described above indicate that CNTO 95 can inhibit cell adhesionand migration. Therefore, we questioned whether this antibody couldblock tumor cell invasion, a multistep process that involves celladhesion, degradation of the matrix, and migration of cells through thedegraded matrix. We chose fibrin as a matrix for tumor cells becauseCNTO 95 was able to block tumor cell adhesion to fibrin (FIG. 3). Asshown in FIG. 10, invasion of A375S.2 cells could be inhibited by LM609,suggesting the involvement of at least αvβ3 in this process. CNTO 95dose dependently inhibited tumor cell invasion through fibrin.Irrelevant IgG and a mAb directed to platelet GPIIb/IIIa (10E5) servedas negative controls. Collectively, these data suggest that CNTO 95 caneffectively block invasion of human melanoma cells.

Invasion of A375S.2 cells through a fibrin gel (5 mg/ml). Invasion assaywas allowed to proceed for 24 h and data was captured as described inMethods. Photomicrographs are representative fields (4× objective lens)of cell invasion in FIG. 21(A) the absence of antibodies, FIG. 21(B)CNTO 95 (10 μg/ml), FIGS. 21(C) and (D) are graphical representation ofcell invasion in presence of CNTO 95, 10E5 F(ab′)₂, LM609, P1F6, LM-P1F6(LM609+P1F6), human and mouse IgGs (H-IgG and M-IgG). Graph FIG. 21(D):The concentration of all antibodies and proteins is 10 μg/ml. The datawere normalized to percent of control (no antibody) which was consideredas 100%, and each point is the mean of three transwell filters (+/−SD).

Conclusion

Cell adhesion, migration and invasion requires integrins such as αvβ3and αvβ5. CNTO 95 is able to functionally block αvβ3 and αvβ5 integrinsthat are expressed by endothelial and tumor cells. CNTO 95 was able toblock migration and invasion of cells that were stimulated by bFGF orserum. These results suggest that the CNTO 95 is a potent inhibitor oftumor and endothelial cell expressed αvβ3 and αvβ5 integrins.

References

-   1. Taylor, L. D., C. E. Carmack, D. Huszar, K. M. Higgins, R.    Mashayekh, G. Sequar, S. R. Schramm, C-C. Kuo, S. L.    O'Donnell, R. M. Kay, C. S. Woodhouse, and N. Lonberg. 1993. Human    immunoglobulin transgenes undergo rearrangement, somatic mutation    and class switching in mice that lack endogenous IgM. International    Immunology 6:579-591.-   2. Lonberg, N., L. D. Taylor, F. A. Harding, M. Trounstine, K. M.    Higgins, S. R. Schramm, C-C. Kuo. R. Mashayekh, K. Wymore, J. G.    McCabe, D. Munoz-O'Regan, S. L. O'Donnell, E. S. G. Lapachet, T.    Bengoechea, D. M. Fishwild, C. E. Carmack, R. M. Kay, and D.    Huszar. 1994. Antigen-specific human antibodies from mice comprising    four distinct genetic modifications. Nature 368:856-859.-   3. Neuberger, M. 1996. Generating high-avidity human Mabs in mice.    Nature Biotechnology 14:826.-   4. Fishwild, D. M., S. L. O'Donnell, T. Bengoechea, D. V. Hudson, F.    Harding, S. L. Bernhard, D. Jones, R. M. Kay, K. M. Higgins, S. R.    Schramm, and N. Lonberg. 1996. High-avidity human IgG monoclonal    antibodies from a novel strain of minilocus transgenic mice. Nature    Biotechnology 14:845-851.-   5. Gastl, G., T. Hermann, M. Steurer, J. Zmija, E. Gunsilius, C.    Unger, and A. Kraft. 1997. Angiogenesis as a Target for Tumor    Treatment. Oncology 54: 177-184.-   6. Eliceiri, B. P., and D. A. Cheresh. 1999. The role of αV    integrins during angiogenesis: insights into potential mechanisms of    action and clinical development. The Journal of Clinical    Investigation 103: 1227-1230.-   7. Friedlander M., P. C. Brooks, R. W. Shaffer, C. M. Kincaid, J. A.    Varner, and D. A. Cheresh. 1995. Definition of two angiogenic    pathways by distinct αV integrins. Science 270: 1500-1502.

Example 6 Production and Characterization of Antibodies to the VariableRegion of CNTO 95

Anti-anti-bodies were prepared by immunization of Balb/c mice with CNTO95. Initial titers from mice immunized with CNTO 95 ranged from a highof >1:40,000, to a low of 1:20,000.

For fusions C371idD, C371idH, C371idI and C371idJ, mice #2, 11, 12 and14 respectively, were IV boosted with 50 mg of CNTO 95 diluted to 100 mLin phosphate buffered saline (PBS). For fusions C371idK and C371idL,mice #16 and 17 respectively, were boosted using 50 mg CNTO 95-mousealbumin conjugate as above. Three days after the IV injection, the micewere sacrificed by cervical dislocation and the spleen was removedaseptically, splenocytes were isolated, and fused to the non-secretingmouse myeloma fusion partner, P3×63 Ag 8.653.

From six fusions utilizing CNTO 95 immunized Balb/c mice, seventeenanti-variable region antibody-producing hybridomas were identified basedon their specific reactivity with the variable region of the human aVb3and aVb5 antibody, CNTO 95 (IgG1k), and for their nonrecognition ofisotypic antigenic determinants.

TABLE 2 Characterization of CNTO 95 Anti-variable region Mabs Anti-IDAnti-ID Inhibition Inhibition Inhibition Mab Mab of ID of ID of IDbinding to inhibition of binding in binding in binding in CNTO 95 CNTO95 the the the Murine Prebound to binding to presence of presence ofpresence of Isotype αVβ3 αVβ3 0.5% NHS 5% NHS 50% NHS C508 IgG2b κ − + −− − C577 IgG1 κ − + − − inhibition C580 IgG1 κ − − − − − C581 IgG1 κ − +− − − C582 IgG1 κ − + − − − C583 IgG1 κ − + − − − C571 IgG1 κ − − − − −C578 IgG2b κ + − − − − C585 IgG2b κ − + − − − C572 IgG1 κ − − − − − C573IgG1 κ − − − − − C574 IgG1 κ − + − − − C575 IgG1 κ − − − − − C576 IgG1 κ− + − − − C579 IgG1 κ − + − − − C584 IgG1 κ − + − − − C586 IgG1 κ − + −− − (NHS = Normal Human Serum)

Seventeen monoclonal antibodies were made to the variable region of CNTO95. Six of them (C571-3, C575, C578 and C580) were demonstrated not toblock binding of the antibody to alpha-V-beta3. The remaining eleven(C508, C576-7, C581-6, C574 and C579) appear to block the active site ofCNTO 95 and inhibit the binding of CNTO 95 to human integrin aVb3 and donot bind to CNTO 95 prebound to its receptor. The non-blocking Mab,C578, can detect CNTO 95 pre-bound to αVβ3. Pooled human serum does notinterfere with the binding of 16 of 17 CNTO 95 anti-variable regionMabs.

These seventeen Mabs could prove useful in pharmacokinetic orimmunohistochemical detection of CNTO 95 in patient and animal tissue orsera samples.

Example 7 Demonstration of Anti-Alpha-V Subunit Antibody Binding toAlpha-V Beta-6 On the Surfaces of Cells

CNTO 95 is capable of recognizing the alpha-V (or alpha5) subunit as itis presented on cell surfaces when complexed with the beta subunitsdesignated beta-III (beta-3) and beta-5. It is important to ascertainthat the epitope for binding the alpha-V subunit is still available whenalpha-V is present in other heterodimeric forms of integrins such asalpha-V, beta-1 or alpha-V, beta-6.

The following study confirms that CNTO 95 has the capability to bind theheterodimeric receptor alpha-5, beta-6 as it occurs on the surface of acell.

Materials and Methods

CNTO 95 antibody was from Centocor (Malvern, Pa. 19355). All otherantibody reagents listed below were purchased from Chemicon,International, Inc. (Temecula Calif.). The control and comparatorantibodies included: H IgG 557276, MAB 1959 to beta1, MAB 2076Z tobeta6, MAB 1953Z to alpha-V, MAB 1976Z to alpha-V-beta-3, MAB 1961Z toalpha-V-beta-5, MAB 2077Z to alpha-V-beta6, MAB 2075Z to beta6, and MAB2074Z to alpha-V-beta6.

HEK-293 cells (Human embryonal kidney cells, ATCC CRC-1573) weretransfected with cDNA constructs to overexpress either human av, b6, oravb6 integrins.

Cell Staining and Flow Cytometric Analysis:

Cell suspensions were prepared by trypsinizing adherent cell cultures,washing and resuspending the cells in serum free media (SFM).Thereafter, the cells were reacted with primary antibody, washed andreacted with a second antibody carrying a fluorescent marker.

Primary antibody reaction: cells (1×10⁶ cells/ml) were resuspended in200 microL SFM, and 2 μL of antibody was added to give a final antibodyconcentration 10 microgm/ml in each tube. Cells were incubated from 45minutes to 1 hour on ice, keeping tubes in the dark. To wash away extraantibody, 3 ml of DPBS was added, and tubes were centrifuged at 1300 RPMfor 3 minutes at 4° C.

Secondary antibody: Phycoerythrin-conjugated secondary antibodies wereadded as described above. After 1 hour, tubes were centrifuged at 1300RPM for 3 minutes at 4° C., and cells were resuspended in 0.5 ml of FACSbuffer.

Flow cytometry: Samples were mixed thoroughly before analysis. Flowcytometric analysis was performed on a Becton-Dickinson FACSCalibur,using both green (FITC) and red (phycoerythrin) channels.

Results

As seen in the upper row of FIG. 22A, mock transfected HEK 293 cellsdemonstrated immunoreactivity for integrin alpha-V, and someimmunoreactivity for beta-6 and avb6 (weak), as shown by comparing theposition of the open curve (control stain) against the shaded curve(test antibody). Transfection of HEK 293 cells with either b6 or avb6cDNA caused a high level of immunoreactivity for avb6 integrin asdemonstrated by comparing the shaded curves in the third panels of rows3 and 4 against the shaded curve in the third panel of row 1. A strongershift toward the right indicates stronger immunoreactivity and higherprotein expression.

Transfection with avb6 did not cause a change in expression of avb3(column 1), avb5 (column 2), or b1 (column 3) integrins compared to mocktransfected cells (FIG. 22B). The positions of the shaded curves in eachof the panels in the lower row (avb6 transfected) are nearly identicalto the positions of the corresponding shaded curves in the upper row(mock transfected). Therefore, any change seen in CNTO 95 binding wouldnot be due to changes in avb3 or avb5 expression.

As shown in FIG. 22C, overexpression of human integrin subunits αV(panel 2) or b6 (panel 3) alone caused a small increase in CNTO 95immunoreactivity, which is indicated by a shift toward the right of theshaded curves compared to panel 1. Transfection with the heterodimerichuman avb6 integrin (panel 4) caused a dramatic increase in CNTO 95staining as demonstrated by a large proportion of the shaded curve tothe right of the vertical line. As a further confirmation, cells weredouble-stained for both avb6 and CNTO 95 (FIG. 22D). As seen previously,mock transfected cells showed a small amount of immunoreactivity foravb6 (panel A) and substantial immunoreactivity for CNTO 95 (FIG. 22Dpanel B). Plotting immunoreactivity of avb6 (vertical axis) against CNTO95 (horizontal axis), a large proportion of mock transfected cells fellinto the lower-right quadrant (FIG. 22D, panel C), indicatingimmunoreactivity to CNTO 95 alone. Analysis of avb6 transfected cellsshowed a large increase in immunoreactivity for avb6 (FIG. 22D panel D),as well as for CNTO 95 (FIG. 22D panel E). Plotting immunoreactivity ofavb6 (vertical axis) against CNTO 95 (horizontal axis) revealed a strongshift in the population of cells toward the upper-right quadrant (FIG.22D panel F), indicating that cells which stained intensely for avb6also stained intensely for CNTO 95. Taken together, the results indicatethat CNTO 95 binds to alphavbeta6 integrin.

Example 8 Characterization of CNTO 95 Ligands in Human Placental Tissue

Human placenta is a source of a spectrum of adhesion molecules includingknown integrins. Ligands binding CNTO 95 from a human placental extractwere identified using commercially available Mabs with knownspecificity.

Human placenta was donated with consent. Approximately 300 g tissue waswashed with ice cold buffered saline followed by addition of 600 ml ofextraction buffer (TBS, pH7.5, 1 mM CaCl₂, 1 mM MnCl₂, 100 mMOctylglucoside (OTG), and EDTA-free protease inhibitor tablets fromRoche Applied Sciences). The tissue was minced with scissors and thenhomogenized using a blender. After homogenation, 17.52 g OTG was addedto the homogenate and the mixture was rotated at 4° C. overnight. Theextract was the supernatant taken after centrifugation in a SORVALLRC5CPLUS centrifuge in a SLA 3000 rotor at 10,000 rpm for 1 hour at 4°C. Extracts were stored at 4° C.

CNTO 95 (Centocor, Malvern, Pa.) and MAB1978 (anti-integrin alphaVpurchased from Chemicon, Temecula, Calif. as ascites fluid and purifiedusing an immobilized protein A (Pierce Chemicals) were coupled toCNBr-activated sepharose 4 Fast Flow (Amersham) according to standardprocedures.

Placenta extracts were incubated with CNTO 95- or MAB1978-conjugatedresin overnight at 4° C. Resins were then loaded to empty columns.Columns were washed with 10 ml of column wash buffer I followed by 10 mlof column wash buffer II. Integrin fractions were eluted with 10 ml ofelution buffer from the columns. The eluted materials were concentratedand stored at 4° C. for further analysis.

Western blot. The integrin fraction were separated by electrophoresis on4-12% SDS polyacrylamide gels and then transferred to nitrocellulosefilters. The filters were blocked with 5% nonfat dry milk in TBScontaining 0.05% Tween 20 (wash buffer) at room temperature for 1 hourand then incubated with anti-integrin antibodies (anti-a5 (P-19) SantaCruz, goat polyclonal IgG 1:500 dilution; anti-aIIb, Chemicon, mousemonoclonal IgG1, 1:1000 dilution; anti-αV (Q-20), Santa Cruz, goatpolyclonal IgG, 1:500 dilution; anti-b1 (4B7R), Santa Cruz, mousemonoclonal IgG1, 1:500 dilution; anti-b1 (N-20), Santa Cruz, goatpolyclonal IgG, 1:500 dilution; anti-β3 (H-96), Santa Cruz, rabbitpolyclonal IgG, 1:500 dilution; anti-β5 (H-96), Santa Cruz, rabbitpolyclonal IgG, 1:500 dilution; anti-β6 (H-110), Santa Cruz, rabbitpolyclonal IgG, 1:250 dilution). After thorough washing, filters wereincubated with appropriate peroxidase-conjugated secondary antibodies(1:20000 dilution). The antigen-antibody complexes were visualized usingSuperSignal West Pico Chemiluminescent Substrate kit (Pierce).

To identify which integrins eluted fraction from CNTO 95 affinity columnWestern blot analysis was performed on strips of the blot incubated withindividual antibody preparations. The strips labeled positively withanti-integrin-aV, -b1, -b3, -b5, and -b6, demonstrating that the aboveintegrin subunits are components of integrin complexes bound by CNTO 95.Since integrins are subunits of alpha subunits and beta subunits, theresults indicate that CNTO 95 binds aVb1, aVb3, aVb5, and aVb6 subunitintegrins.

To further define the integrin specificity of CNTO 95, integrinfractions purified from CNTO 95 affinity column were examined by Westernblots with antibodies against integrin a5 and integrin aIIb, it shouldbe noted that placenta contains abundant integrin a5b1 and integrinaIIbb3. Purified integrin a5b1 and crude placenta extract, were stainedby anti-integrin a5 or anti-aIIb antibody. However, there were nodetectable signals for a5 and aIIb in lanes loaded with proteinspurified by CNTO 95 affinity chromatography.

Therefore, CNTO 95 binds all subunit of the integrins containing alphaVtested and not a5 or aIIb containing heterodimers.

Example 9 In Vitro Angiogenesis: Microvessel Sprouting

Rat aortic ring assay. An early event during tumor-induced angiogenesisis the formation of microvessel sprouts from established blood vesselsthat migrate towards the tumor. A microvessel sprouting assay was usedto test the in vitro anti-angiogenic activity of CNTO 95. In this assay,freshly excised rat aortas were cultured in 3-dimensional fibrin orcollagen gels. In the presence of various angiogenic factors such asbFGF, the aorta sprouts microcapillaries after a few days. This processis a representation of angiogenesis, as it involves endothelial celladhesion, migration, invasion, and proliferation.

The rat aortic ring assay was performed as described by Nicosia et al.(Lab Invest. 63: 115-122, 1990) with slight modifications (Sassoli, etal. Thromb Haemost, 85: 896-902, 2001). 10 mm diameter agarose wells(1.5%) prepared in 100×15 mm tissue culture dishes were filled with M199media containing rat tail type 1 collagen (2.8 mg/ml, Becton Dickinson),NaHCO3 (28 mM) and either CNTO 95 (10 ug/ml), nonspecific control murineIgG (20 mg/ml) or BSA (20 mg/ml). Rat aortic ring sections (1 mm) wereplaced on top of collagen gels within the agarose rings; wells werefilled with collagen solution and incubated at 37° C. After the collagenhad gelled, the collagen-aortic ring sandwiches were transferred to 12well plates containing 1 ml EBM-2 medium, BSA (0.1%), bFGF (10 ng/ml),penicillin (100 U/ml), streptomycin (100 mg/ml), amphotericin B (0.25mg/ml) (all from Clonetics), and either CNTO 95 (increasingconcentrations), non-specific control mIgG (20 ug/ml), or BSA (20mg/ml). Plates were maintained in a 37° C. tissue culture incubator withmedia changes every other day. After 10 days, the number and length ofmicrovessel sprouts originating from each aortic ring was quantifiedmicroscopically using the Phase 3 Image Analysis System.

As shown in FIG. 23, CNTO 95 inhibited sprouting of microvessels fromaortas excised from rats in a dose-dependent manner These resultsdemonstrate that CNTO 95 is an inhibitor of angiogenesis in vitro.

Example 10 Anti-Alpha V Antibody Blocks Angiogenesis In Vivo

Rat and Monkey Matrigel Assays

In order to determine whether CNTO 95 could functionally block aVb3 andaVb5 integrins, a non-human primate model and a nude rat model of growthfactor-induced angiogenesis and in a nude rat model of tumorcell-induced angiogenesis were used. The monkey study was performed atCharles River Laboratories (Worcester, Mass.) on young adult femaleCynomolgus monkeys (species macaca fascicularis). Nude female rats (5-7weeks old) were obtained from Harlan (Indianapolis, Ind.). Recombinanthuman bFGF was obtained from R&D Systems. Matrigel, prepared from theEngelbreth-Holm-Swarm tumor, was obtained from Becton Dickinson.

Liquid Matrigel was maintained at 4° C. The angiogenesis assays wereperformed as described (Trikha, et al. Cancer Res., 62: 2824-2833,2002). For growth factor induced angiogenesis, human bFGF (5 mg/ml) wasadded to the Matrigel solution and allowed to mix thoroughly overnight.The Matrigel was then mixed with antibodies or control solutions andkept on ice. For tumor-cell induced angiogenesis, human melanoma M21cells were harvested with trypsin-EDTA, centrifuged, washed twice withDMEM, and resuspended in ice-cold DMEM. M21 cells were gently added tothe Matrigel solution at a final concentration of 0.5×10⁶/ml. The tumorcell-Matrigel solution was gently mixed and stored on ice until it wasinjected into nude rats.

Monkeys were injected at each site subcutaneously with 2 ml of Matrigelsolution, while rats were injected with 1 ml of Matrigel each. In thetumor cell-induced study, rats were injected at two sites with 1 ml ofthe ice-cold tumor cell-Matrigel solution. Gel formation was confirmedafter injection. Animals received test article via intravenous orintraperitoneal bolus injection. At the end of each study, animals wereeuthanized and Matrigel harvested from the injection sites. Matrigelimplants were weighed, photographed and graded for angiogenesis usingthe Phase 3 Image Analysis System. To measure the total area ofneovessels, photomicrographs were taken from both the top surface andthe bottom surface of each Matrigel plug at 2× magnification on theinverted phase contrast microscope. The vessel length and number ofvessels per field were calculated using the tracing function within thePhase 3 Image System. The mean value from all 2× fields was calculatedfor each Matrigel plug, and the mean vessel number and vessel length foreach test group was calculated.

As a third method to measure angiogenesis, immunohistochemistry wasperformed on 10 mm serial cryostat sections cut from frozen Matrigelplugs. Sections were immediately fixed in cold acetone (5 min) andair-dried. The sections were washed 3 times in PBS to remove frozenmounting media, blocked for 1 hour with 5% mouse serum and 5% goat serumin PBS and rinsed in PBS. The sections were then blocked withAvidin-Biotin solution (X0590, DAKO Corporation, Carpinteria, Calif.)for 10 min. After washing, endogenous peroxidase was quenched byincubation in 3% hydrogen peroxide for 10 min. Then, the sections wereincubated for 60 min with primary antibody (mouse anti-human PECAM, BDPharMingen, Bedford, Mass.; 10 mg/ml) diluted with DAKO antibody diluentsolution (S3022, DAKO). Immunoreactive sites were detected using a DAKOKit, and sections were counterstained with hematoxylin. An irrelevantmouse IgG1 was used as a negative control in all cases. Photomicrographswere taken from all slides at 20× magnification; each entire section wasphotographed. The vessel density per field was calculated using thePhase 3 Image System software. Vessel density was quantitated bymeasuring the percentage of cross-sectional area of each Matrigelsection occupied by stained microvessels. The mean value for each slidewas calculated, and the mean vessel density for each group wasdetermined.

Results

Inclusion of human bFGF in Matrigel implants in monkeys and ratsresulted in increased angiogenesis as measured by vessel length, number,and vessel density (FIG. 24-26). Systemic treatment of rats with CNTO 95significantly inhibited bFGF-stimulated increases in vessel length andtotal vessel number within Matrigel implants as measured by visualinspection. Inhibition of angiogenesis by CNTO 95 was dose-dependent,with a dose of 1 mg/kg being active in this model (FIG. 24). Results ofthe immunostaining of vessels with anti-CD31 were consistent with thoseobtained by direct visual counting of microvessels.

Subcutaneous injection of Matrigel containing human bFGF in cynomolgusmonkeys resulted in increased angiogenesis as measured by vessel length,number, and vessel density (FIG. 25A-C). Systemic treatment of monkeyswith CNTO 95 significantly inhibited bFGF-stimulated increases in vessellength (FIG. 25A) and total vessel number (FIG. 25B) within Matrigelimplants as measured by image analysis quantification. Systemictreatment of monkeys with CNTO 95 also reduced the bFGF-stimulatedincrease in microvessel density within Matrigel implants as measured byimmunostaining for CD31 expression and image analysis (FIG. 25C).

In the tumor cell-induced angiogenesis model, a single dose of 10 mg/kgof CNTO 95 inhibited tumor cell-induced angiogenesis. No difference ininhibitory activity was observed when CNTO 95 was administered as asingle intravenous dose or when it was mixed with the Matrigel-tumorcell suspension prior to injection into the rats. Inhibition ofangiogenesis by CNTO 95 was demonstrated by decrease in the length andnumber of microvessels (FIG. 26) and decrease in hemoglobin content inthe Matrigel plugs (data not shown).

Collectively, these results indicated that CNTO 95 inhibitedangiogenesis stimulated by either bFGF in both nude rats and non-humanprimates. CNTO 95 also prevents human tumor cell induced angiogenesis inan immunosuppressed animal.

Example 11 Anti-Tumor Effect of Anti-Alpha V Antibody in Nude Mice andRats

For studies performed in nude mice, female nude mice, aged 4-5 weeks,were purchased from Charles River Laboratories (Wilmington, Mass.), andwere maintained according to the NIH standards established in the‘Guidelines for the Care and Use of Experimental Animals’. Twenty micewere inoculated subcutaneously with A375.S2 cells (3×106) in the flankregion (day 0). On day 3, the mice were randomly divided into twogroups. One group was injected i.p. with CNTO 95 (10 mg/kg in PBS),while the other group received vehicle. Dosing was continued three timesa week thereafter until day 26. Tumors were measured by calipers twice aweek, and tumor volumes were calculated by the formula(length×width2/2). Body weights were also recorded weekly.

For studies performed in nude rats, female nude rats, aged 6-7 weeks,were purchased from Harlan (Indianapolis, Ind.), and were maintainedaccording to the NIH standards established in the ‘Guidelines for theCare and Use of Experimental Animals’. Twenty rats were inoculatedsubcutaneously with A375.S2 cells (3×106) in the flank region (day 1).On day 4, the rats were randomly assigned to two groups. One group wasinjected i.v. with CNTO 95 (10 mg/kg in PBS), while the other groupreceived an isotype-matched control IgG (10 mg/kg). Dosing was continuedweekly thereafter until day 46 (total of 6 doses). Tumors were measuredby calipers twice a week, and tumor volumes were calculated by theformula (length×width2/2). Body weights were also recorded weekly.Statistical comparison of group mean tumor volumes was performed usingStudent's t-test, 2-tailed analysis.

Results

To determine the anti-tumor efficacy of CNTO 95 in vivo, a human A375.S2melanoma xenograft tumor model was established in nude mice. Mice weretreated with CNTO 95 (10 mg/kg) 3 times per week by i.p. injection,starting 3 days after tumor inoculation. As shown in FIG. 27, dosingwith CNTO 95 inhibited growth of human melanoma tumors in nude mice. Atday 26 CNTO 95 inhibited tumor growth by 80% compared to tumors fromcontrol-treated animals. In this model CNTO 95 does not interact withhost angiogenic vessels, since CNTO 95 does not bind mouse integrins,suggesting that blockade of human tumor-expressed integrins alone caninhibit tumor growth in mice independent of anti-angiogenic effects.

To determine the anti-tumor efficacy of CNTO 95 in another xenograftanimal model, an A375.S2 human melanoma model was developed in femalenude rats. In this model CNTO 95 is capable of blocking both ratangiogenic integrins and human tumor cell expressed integrins. Weeklytreatment of tumor-bearing nude rats with CNTO 95 at 10 mg/kg reducedtumor growth compared to the isotype-matched human IgG control mAb (FIG.28). By day 46, treatment with CNTO 95 resulted in a significantreduction in final tumor size compared to control-treated nude rats(P=0.0007).

In addition to blocking integrins on angiogenic endothelium, CNTO 95 hasthe ability to inhibit integrin function on tumor cells themselves.AlphaV integrins have been suggested to play critical roles in tumorcell biology. Therefore, the use of CNTO 95 has applicability tomultiple tumor types with different integrin expression patterns.

In the nude mouse xenograft model, CNTO 95 does not cross-react withhost integrins, however, treatment with CNTO 95 significantly inhibitedthe growth of the αvβ3/β5positive melanoma tumors. In a previous report(Trikha et al. supra), it was demonstrated that antibody against αvβ3 onthe surface of human melanoma cells partially inhibited tumor growth innude mice. In that study, m7E3 F(ab′) 2, a murine antibody which bindsand blocks human αvβ3 and aIIbβ3, directly reduced growth of a humanmelanoma xenograft without blocking host cell integrins. CNTO 95 differsfrom m7E3 F(ab′) 2 in that it is a full-length human IgG that recognizesαvβ3 and αvβ5, but not the aIIbβ3 that is predominantly expressed onplatelets.

Together these data suggest that through combined blockade of αvβ3 andαvβ5 integrins on tumor and endothelial cells, CNTO 95 may have multiplemechanisms of action that contribute to its observed antitumor efficacyin animal models.

It is becoming increasingly clear that although targeted therapy holdsgreat promise, combination drug regimens will likely be necessary foroptimal efficacy. CNTO 95 by itself targets multiple crucial receptorsinvolved in tumor growth, angiogenesis and metastasis. An additionaladvantage of CNTO 95 is its fully-human nature, which will allowlong-term and repeated use with anticipated safety due to lack of theHAMA reactions seen with murine antibodies.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

1. An isolated human anti-αV subunit antibody or antigen-bindingfragment thereof, comprising a human heavy chain having CDR1, CDR2 andCDR3 regions comprising the amino acid sequences of SEQ ID NO:1, SEQ IDNO:2 and SEQ ID NO:3 respectively, and a human light chain having CDR1,CDR2 and CDR3 regions comprising the amino acid sequences of SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
 2. The isolated antibodyor antigen-binding fragment thereof of claim 1, which binds to human αVintegrin subunit with a Kd of 10⁻⁸ M or less.
 3. An isolated humanmonoclonal antibody or antigen-binding fragment thereof that competesfor binding to human αV integrin subunit with the antibody of claim 1.4. The antibody or antigen-binding fragment thereof of claim 1 whichcompletely inhibits M21 cell adhesion to vitronectin.
 5. The antibody orantigen-binding fragment thereof of claim 1, comprising a human IgGheavy chain and a human kappa light chain.
 6. The antibody orantigen-binding fragment thereof of claim 1, comprising an IgG1 or IgG3heavy chain.
 7. A pharmaceutical composition comprising the antibody orantigen-binding fragment thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 8. A composition according to claim 7, furthercomprising an effective amount of at least one compound or proteinselected from at least one of a detectable label or reporter, ananti-neoplastic agent, a TNF antagonist, an antirheumatic, a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial, an antipsoriatic, acorticosteroid, an anabolic steroid, an erythropoietin, animmunoglobulin, an immunosuppressive, a growth hormone, a hormoneantagonist, a reproductive hormone antagonist, a hormone releasemodulator, a hormone replacement drug, an antidepressant, anantipsychotic, a stimulant, an asthma medication, a beta agonist, aninhaled steroid, an epinephrine or analog, a cytokine, or a cytokineantagonist.
 9. A composition according to claim 7, wherein the antibodyor antigen-binding fragment thereof is combined with an anti-neoplasticagent selected from a radiopharmaceutical, an estrogen receptormodulator, a retinoid, a topoisomererase inhibitor, a cytotoxin, analkylating agent, a nitrogen mustard, a nitrosourea, an antimetabolite,a mitotic inhibitor, and a radiosensitizer.
 10. A composition accordingto claim 9, wherein the anti-neoplastic agent is dacarbazine.
 11. Animmunoconjugate comprising the antibody or antigen-binding fragmentthereof of claim 1 linked to a therapeutic agent.
 12. Theimmunoconjugate of claim 11 wherein the therapeutic agent is acytotoxin.
 13. The immunoconjugate of claim 11 wherein the therapeuticagent is a radioisotope.
 14. A pharmaceutical composition comprising theimmunoconjugate of any one of claims 11-13 and a pharmaceuticallyacceptable carrier.
 15. An article of manufacture for humanpharmaceutical or diagnostic use, comprising packaging material and acontainer comprising a solution or a lyophilized form of at least oneisolated human anti-αV subunit antibody according to claim
 1. 16. Thearticle of manufacture of claim 15, wherein said container is acomponent of a parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.
 17. The antibody or antigen-binding fragment of claim1, which is of immunoglobulin class IgG1, IgG2, IgG3, IgG4 IgA, IgD IgEor IgM.
 18. The antigen-binding fragment of claim 1 selected from thegroup consisting of Fab, Fab′, F(ab′)₂ and Fv.
 19. The human anti-αVsubunit antibody or antigen-binding fragment thereof of claim 1, whichis produced recombinantly.
 20. The human anti-αV subunit antibody orantigen-binding fragment thereof of claim 1, which is produced bychemical synthesis.
 21. The human anti-αV subunit antibody orantigen-binding fragment thereof of claim 1, which is produced by usinga hybridoma.